Mystery Rays from Outer Space

A couple weeks ago I was having a chat with a friend about cancer immunity (as one so often does) and he asked if the Holy Grail of cancer immunity would be to identify tumor antigens. Not at all. There are hundreds of tumor antigens known. (The journal Cancer Immunity hosts a database that lists many of the known ones.) The problem is if anything the opposite; there are too many antigens, and many are one-offs, unique to one or a handful of tumors and of no use to most patients. A better Holy Grail would be a single target that many tumors have in common.

Our genomes are littered with the withered corpses of ancient retroviruses. Everyone has them. These human endogenous retroviruses (HERVs) are defective, and their proteins are usually not expressed, or are expressed at low levels. Because they’re not normally expressed much, they don’t necessarily tolerize the immune system. At least hypothetically, if there are pathologic conditions in which HERVs become expressed, they might form targets for immunity.

As it happens, there may be several such conditions. It’s been suggested (though not, to my inexpert eye, all that convincingly) that HERVs might represent targets in autoimmunity. More usefully, Douglas Nixon’s group showed some evidence, last fall, that HIV infection upregulates HERVs, offering a target for CTL that (unlike HIV itself) isn’t constantly mutating.¹ And it’s been suggested for quite a while that HERVs might be immunogenic in tumors.

For example, over ten years ago it was shown that patients with certain kinds of tumors, which consistently show high-level HERV activation, often have antibody responses to HERVs.² However, in general, antibodies are not particularly effective against tumors, and as far as I know, nothing much arose directly from the antibody findings.

On the other hand, T cells are (at least sometimes) more effective against tumors; and T cell immunity was linked to HERVs first (as far as I know) in 2002,³ with the observation that a melanoma tumor antigen was derived from a HERV. Some similar work has followed.⁴

So: HERVs are potential antigens; they are more or less immutable; they can be upregulated in some tumors; and they can trigger an immune response by antibodies and by T cells. These are interesting observations, but is this at all relevant for tumor treatment?

The next step in answering that question came out recently, in J Clin Invest. ⁵ Here we see not just reactive T cells (that is, T cells specific for HERV peptides) but a potent immune response that actually cleared a metastatic tumor. The response was due to an allogeneic bone marrow transplant, and when they tracked down the target peptide for the immune response, it was directed against a HERV peptide:

The genes encoding this antigen were found to be derived from human endogenous retrovirus (HERV) type E and were expressed in RCC cell lines and fresh RCC tissue but not in normal kidney or other tissues.

It’s still far from clear how universal a target HERVs might be. This group identified a HERV target in one of their patients, but they treated 74 patients, saw at least partial responses in 29 of those patients, sought to identify targets in four of the responders, and found the HERV target in just one of the four. Some of the other targets were apparently the more standard mutated proteins, specific to the individual tumor.

This peptide target, by the way, is from a group E HERV; most of the previous work has focused on group K HERVs, which tend to be more active and are expressed to some extent in normal tissue. HERV-E generally are pretty quiescent, so if tumors do upregulate HERV-E, it would be a more specific target. The authors did check, and found that most of that particular type of tumor expressed HERV-E. Interestingly, this is the kind of tumor that is most likely to be responsive to immunotherapy:

A histological review of the RCC⁶ cell lines and fresh RCC tissues used in experiments presented in this article showed all to be clear-cell carcinomas, with more than half expressing HERV-E transcripts. Furthermore, limited preliminary data from an ongoing study of fresh tumors suggest that this HERV-E may have transcriptional activity limited to the clear-cell variant of kidney cancer (unpublished observations), which is intriguing given the track record for this tumor being the immunoresponsive subtype of RCC.

It would be a very useful discovery if this turns out to be a common antigen among these tumors. That said, there are some other known common tumor antigens — such as tyrosinase in melanomas — and immunization hasn’t proven a silver bullet in those yet. But it’s early days, still.

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1. Garrison, K. E., Jones, R. B., Meiklejohn, D. A., Anwar, N., Ndhlovu, L. C., Chapman, J. M., Erickson, A. L., Agrawal, A., Spotts, G., Hecht, F. M., Rakoff-Nahoum, S., Lenz, J., Ostrowski, M. A., and Nixon, D. F. (2007). T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection. PLoS Pathog 3, e165. [↩]
2. Boller, K., Janssen, O., Schuldes, H., Tonjes, R. R., and Kurth, R. (1997). Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K. J Virol 71, 4581-4588.[↩]
3. Schiavetti, F., Thonnard, J., Colau, D., Boon, T., and Coulie, P. G. (2002). A human endogenous retroviral sequence encoding an antigen recognized on melanoma by cytolytic T lymphocytes. Cancer Res 62, 5510-5516.[↩]
4. Rakoff-Nahoum, S., Kuebler, P. J., Heymann, J. J., E Sheehy, M., M Ortiz, G., S Ogg, G., Barbour, J. D., Lenz, J., Steinfeld, A. D., and Nixon, D. F. (2006). Detection of T lymphocytes specific for human endogenous retrovirus K (HERV-K) in patients with seminoma. AIDS Res Hum Retroviruses 22, 52-56.[↩]
5. Takahashi, Y., Harashima, N., Kajigaya, S., Yokoyama, H., Cherkasova, E., McCoy, J.P., Hanada, K., Mena, O., Kurlander, R., Abdul, T., Srinivasan, R., Lundqvist, A., Malinzak, E., Geller, N., Lerman, M.I., Childs, R.W. (2008). Regression of human kidney cancer following allogeneic stem cell transplantation is associated with recognition of an HERV-E antigen by T cells. Journal of Clinical Investigation DOI: 10.1172/JCI34409[↩]
6. RCC: “Renal cell carcinoma.” IY[↩]

Natural killer cells were originally identified as cells that spontaneously killed cancer cells. It’s been a bit surprising, then, that there’s been relatively little direct evidence that NK cells protect against spontaneous cancer.

For example, there was the study I talked about some time ago, looking at tumors in equilibrium with the immune system. There, the authors treated mice with a  carcinogen, waited until tumors stopped arising, and immunosuppressed the tumor-free survivors. ¹ In most of these apparently cancer-free animals, immune suppression resulted in tumors appearing within a few weeks. This showed that the immune system can control tumors.

The interesting part (at least, the interesting part as far as NK cells are concerned) was that treatment with anti-NKG2D did not let tumors grow out; whereas shutting down T cells (with anti-CD4/anti-CD8) or interferon (with anti-IFN) did let the tumors reappear. NKG2D is an important receptor for NK cells, so the implication was that NK cells were not keeping the tumor in check, but T cells were.

Now, however, a similar set of experiments has shown that’s not necessarily true — NK cells probably are important in controlling some tumors. The paper is
Guerra, N., Tan, Y. X., Joncker, N. T., Choy, A., Gallardo, F., Xiong, N., Knoblaugh, S., Cado, D., Greenberg, N. R., and Raulet, D. H. (2008). NKG2D-Deficient Mice Are Defective in Tumor Surveillance in Models of Spontaneous Malignancy. Immunity 28, 571-580. DOI: 10.1016/j.immuni.2008.02.016

David Raulet’s group has been one of the leaders in NK cell research, and as far as I know they are the first to have made a knockout mouse lacking NKG2D. The mice are normal and happy and actually have normal numbers of NK cells that are functional. NK cells notoriously have many ligands, which is one of the reasons it took longer to figure out NK receptor/ligand interactions than for T cells, and presumably there are enough alternatives to NKG2D that NK cells can get whatever signals they need during development. However, of course, none of the ligands for NKG2D triggered NK cells.

Rather than wait for truly spontaneous tumors (which are rare enough even in mice that you need very large numbers of mice to figure out what’s going on) they crossed the NKG2D -/-mice with a couple of transgenic lines that are highly cancer-prone. They also tried treating the mice with carcinogens, as was done in the Koebel et al study I mentioned earlier.

The carcinogen-treated NKG2D knockout mice got no more tumors than did wild-type mice — so that’s exactly consistent with the previous experiment. However, the “spontaneous” tumor transgenic models showed a big difference. The NKG2D knockouts had much earlier, more aggressive tumors than did the wild-type mice.

As well as evidence for cancer equilibrium, the Koebel et al paper showed evidence for immunoediting. ² That is, tumors that grow in the presence of a healthy immune system are resistant to the immune response — they have been selected for immune invisibility or resistance. By comparison, tumors that grow in immune deficient mice are much more immunogenic — they haven’t had to develop immune resistance.

In one of the two transgenic systems, the NKG2D knockout mice showed the same thing: Their tumors were more likely to have NKG2D ligands than the wild-type mice. “These data suggest that NKG2D-dependent immunoselection (or editing) favors loss of NKG2D ligands on early-arising, aggressive tumors.”

But in the other tumor system no such evidence was seen; NKG2D ligands were just as prevalent:

There was no indication in this survey that expression of NKG2D ligands was selected against in Klrk1+/+ mice, despite the clear evidence that NKG2D-mediated surveillance is operative for these lymphomas. These data suggest that evasion of NKG2D-mediated surveillance by Eμ-myc-induced lymphomas occurs by mechanisms that do not depend on loss of NKG2D ligands.

So it seems that NK immune surveillance is much more complicated than the (already very complicated) T cell immune surveillance of tumors:

Taken together, these data suggest that the role of NKG2D-dependent surveillance differs in the three types of tumors studied here. In the case of early-arising prostate carcinomas in TRAMP mice, many of the tumors are eliminated, and the few that are not eliminated evade surveillance by extinguishing expression of NKG2D ligands. In the case of Eμ-myc lymphomas, it appears that the emerging tumors are mostly NKG2D sensitive, but a fraction of tumors escape NKG2D surveillance without losing NKG2D ligands. … The final category is represented by late-arising prostate carcinomas, which appear to be generally refractory to NKG2D-dependent surveillance.

But the bottom line is that NK cells do control some tumors. That’s not a surprise, because it’s pretty much been the assumption for a long time, but it’s reassuring to get evidence for it.

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1. Koebel, C. M., Vermi, W., Swann, J. B., Zerafa, N., Rodig, S. J., Old, L. J., Smyth, M. J., and Schreiber, R. D. (2007). Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903-907.[↩]
2. Of course, this wasn’t the first evidence for immunoediting.[↩]

Breart et al, Fig. 3. Direct action of CTLs on individual tumor cells drives tumor regression

Intravital microscopy — microscopic analysis, in real time, of processes within a living animal — has been used in immunology for maybe a decade now, but it hasn’t lost its cool factor yet. I don’t know that there have been any great intellectual breakthroughs arising from the work, but we have learned a fair bit about, say, interactions between T cells and their targets, and migration patterns, and so on. And of course there’s a huge help in visualization, which undoubtedly helps people understand what’s happening and, hopefully, develop other experimental approaches to test it.

Just as importantly, the “Awesome” factor of these things is absolutely off the scale. I pointed out Uli von Andrian’s collection of intravital videos the other day, and now the latest issue of Journal of Clinical Investigation has a paper from Phillipe Bousso’s group, showing 2-photon microscopy of cytotoxic T lymphocytes attacking a tumor.

The paper is:

Breart, B., Lemaître, F., Celli, S., Bousso, P. (2008). Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. Journal of Clinical Investigation, 118(4), 1390-1397. DOI: 10.1172/JCI34388

They used the EL4/EG7 tumor model in mice. These cells form solid tumors in C57BL/6 mice, and are not rejected by the immune system. The EG7 cells are derived from EL4; they have had a defined antigen introduced, and if you transfer activated T cells against the antigen, the tumor will be rejected. If you transfer naive T cells, and depend on them to be activated by the tumor, you’re out of luck; the tumor is not rejected. ¹ They were able to watch all these things happening, in real time.

Here’s what happens with activated CTL (orange) around a tumor site (tumor cells in yellow/greenish). Watch the CTL zipping around merrily in areas where there are no tumor cells, and then screeching to a halt as they identify tumor antigen, engage their targets, and begin to kill:

(Embedded video! I’m so MySpace! I’m going to use tripple exclamation marks and mispell lot’s of words!!!)

The article is free access, I believe, so you should check it out for yourself; there are five videos to watch in the supplemental data. They show CTL engaging tumor cells and actively killing them, using indicators for cell death so they don’t have to guess what’s happening.

I think this is mainly a technical tour de force, and the amount of new information about tumor immunology is relatively small. But there are a couple things of interest. One is that naive T cells — the guys who do not reject the tumor — seem kind of indifferent to the whole thing. It’s not a question of the CTL entering the tumor, and then being turned off (which would have been my guess); rather, the naive cells never even entered the tumor in the first place:

Although CTL infiltration was quite variable in the different regions of the tumor (Figure 6A), EG7 patches were eliminated in CTL-rich areas, which was evidence that in vivo primed CTLs were not grossly impaired in their ability to kill target cells … Thus, the low level of CD8⁺ T cell infiltration, rather than a defect in the cytotoxic activity, appeared to be responsible for the inefficient response mounted by in vivo primed OT-I T cells.

Another surprising finding — which is so different from previous work in different systems that I’m hesitant to believe it — is the timing of cell killing. Previous studies (such as the von Andrian paper² that produced this video) have suggested that CTL kill their targets in something under an hour; maybe 30 minutes or even less. Here. Bousso’s group find that the tumor cells take something like 6 hours to be killed. That’s such a large difference — and has such important implications for effectiveness of CTL killing — that, as I say, I’d like to see it confirmed before I take it to the bank.

Bousso’s web site has a bunch of other equally fascinating videos; check them all out.

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1. This is probably related to the ability of tumors to suppress immune responses, which I’ve talked about before.[↩]
2. Mempel, T. R., Pittet, M. J., Khazaie, K., Weninger, W., Weissleder, R., von Boehmer, H., and von Andrian, U. H. (2006). Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25, 129-141.[↩]

Chronic inflammation

It’s pretty well-established now that the immune system can, and normally does, protect us against cancer. In particular, the adaptive immune response (especially T cells) clearly limits cancer growth, so that the only cancers we can detect clinically are those that have developed defenses against the adaptive immune response (see here for more).It seems paradoxical, then, that the immune response may also help cancers develop. However, there is a fair bit of evidence that a chronic immune response can actually help drive cancer development. This is especially true for the innate immune system, but long-term stimulation of the adaptive immune system may also be carcinogenic.

The role of the innate system is relatively easy to understand (at least, it made sense to me, which is no guarantee that it makes sense). Chronic inflammation is a bad thing — there are lots of checks built in to the immune response to try to prevent that — and conditions where there’s chronic inflammation are often clearly associated with cancer. The example that jumps to my mind is hepatitis B infection. As far as I know, it’s not generally believed that the virus itself is carcinogenic per se (that is, in contrast to things like Kaposi’s Sarcoma herpesvirus, or some human papillomaviruses, which seem to have the ability to drive infected cells into a de-regulated state). Rather, the increased risk of cancer associated with HBV infection (about five to fifteen times higher than the general population) is probably because of the chronic inflammation that the virus infection causes. ¹

There are a number of ways the chronic inflammation can lead to cancer. Simply increasing cell turnover (as cells are killed by the inflammation and have to be replaced) increases the chance of a dangerous mutation arising. Inflammatory factors can act as growth factors for tumor cells. Reactive oxygen species produced as part of the inflammation may increase mutation frequency. And so on. ²

Inflammation in carcinogenesis

It’s a little more surprising to contend that the adaptive immune system may also help drive cancers. My first response to the concept was to dismiss it, because immune-deficient mice actually have more tumors than wild-type mice, not fewer. However, as I realized within ten seconds of my dismissal, that’s not counterevidence; adaptive immunity could drive cancer at one stage and protect against it at another stage, and the experiments in question would only reveal which of the processes had the larger effect. And in fact, it turns out that although immune-suppressed people (AIDS patients, or transplant recipients) have increased risk of many tumors, they are at reduced risk of other kinds. For example, prostate tumors are less frequent in AIDS patients than in matched controls,³ and breast cancers are less common in transplants recipients⁴

I don’t think the mechanism(s) underlying this are as well understood as for innate immunity (and that itself is still not well understood). It’s likely that adaptive immunity plays a part in establishing some forms of chronic inflammation. In any case, there’s a fair bit of interest in blocking inflammation during cancer as a component of treatment.

It’s worth emphasizing that the great majority of tumors — if they show any change in incidence in immune-suppressed people — are more frequent; it’s just a few types of tumors that are less frequent. Don’t go immune-suppressing yourself in an attempt to avoid cancer.

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1. There are some virus factors that might be more directly correlated with cancer, but the link is rather indirect.[↩]
2. Here’s a nice review:de Visser, K.E., Eichten, A., Coussens, L.M. (2006). Paradoxical roles of the immune system during cancer development. Nature Reviews Cancer, 6(1), 24-37. DOI: 10.1038/nrc1782[↩]
3. Frisch, M., R. J. Biggar, E. A. Engels, and J. J. Goedert. 2001. Association of cancer with AIDS-related immunosuppression in adults. JAMA 285: 1736-1745.[↩]
4. Stewart, T., S. C. Tsai, H. Grayson, R. Henderson, and G. Opelz. 1995. Incidence of de-novo breast cancer in women chronically immunosuppressed after organ transplantation. Lancet 346: 796-798.[↩]

My 4-year-old son has been sick all week, probably with influenza (his doctor diagnosed it as influenza, but didn’t perform any specific tests) so I’ve got a little behind on my work,¹ and today’s post is going to be a little short. I’m intrigued by an article in the latest Immunological Reviews, which I’ll probably post on later today or perhaps tomorrow, depending on how fast my cells are growing; if I need to split them, the blog comes second.

But before I get to that, I want to point out the introduction to the issue, a tumor immunology top ten list by Olivera Finn, which she introduces thus:²

I am often surprised with the unawareness that can be encountered in the scientific and medical community about the great extent of knowledge that has accumulated in tumor immunology.

I’ve talked about a number of the items on her list, so I’m presenting the list both with the references Finn offers and, where possible, my own posts on the subject:

No.10. Tumors express antigens recognized by the immune system; many have been fully characterized.
Graziano DF, Finn OJ. Tumor antigens and tumor antigen discovery. Cancer Treat Res 2005;123:89–111.
I’ve talked about one example of this here

No.9. Tumors are seen as dangerous by the immune system.
Rock KL, Hearn A, Chen CJ, Shi Y. Natural endogenous adjuvants. Springer Semin Immunopathol 2005;26:231–246.
One of my first posts here was about this.

No.8. There is specific and effective immune surveillance of cancer.
Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Annu Rev Immunol 2004;22:329–360.
I have several posts on this, including this one (specifically about the Dunn et al paper, in fact), and others here and here.

No.7. The immune response is an important biomarker in cancer
Finn OJ. Immune response as a biomarker for cancer detection and a lot more. N Engl J Med 2005;353:1288–1290.
I made a rather peripheral mention of this here.

No.6. Immune responses against cancer can be both good and bad
de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 2006;6:24–37.
This is the one I want to talk about.

No.5. Tumors fight back but do not always have to win
Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 2007;25:267–296.
I’ve talked about immune evasion by tumors in several places: Here and here, for example.

No.4. Passive immunotherapy of cancer is effective
June CH. Adoptive T cell therapy for cancer in the clinic. J Clin Invest 2007;117:1466–1476.
Cheson BD. Monoclonal antibody therapy for B-cell malignancies. Semin Oncol 2006;33 (Suppl.):S2–S14.

No.3. Active immunotherapy of cancer (cancer vaccines) is marginally effective and can be improved
Finn OJ. Cancer vaccines: between the idea and the reality. Nat Rev Immunol 2003;3:630–641.

No.2. Combination of immunotherapy and standard therapy is possible
Emens LA, Jaffee EM. Leveraging the activity of tumor vaccines with cytotoxic chemotherapy. Cancer Res 2005;65:8059–8064.

No.1. Cancer immunoprevention is an attainable goal
Lollini PL, Cavallo F, Nanni P, Forni G. Vaccines for tumour prevention. Nat Rev Cancer 2006;6:204–216. 26.

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1. As the butcher said when he backed into his sausage machine[↩]
2. Olivera J. Finn (2008) Tumor immunology top 10 list. Immunological Reviews 222 (1) , 5–8 doi:10.1111/j.1600-065X.2008.00623.x [↩]

A while ago I talked about a paper that demonstrated that cancers establish a long-term equilibrium with the immune system. That paper provided a formal demonstration of a concept that was already widely accepted — that cancers can arise and persist for a long time in balance with the immune system, so that the immune system isn’t able to completely eliminate the cancerous cells, but the tumor is not able to grow or spread, either. Evidence in humans shows that this equilibrium can persist for many years. In some cases, the tumor may eventually mutate to a form that can more completely avoid the immune system, and those tumors become clinically detectable. In other cases, perhaps, the immune system may finally destroy all vestiges of the tumor, and the person won’t ever know that he was carrying the potentially-cancerous cells; or the balance may persist for a lifetime, until the person dies of something altogether different.

There was an interesting implication to that concept that I had overlooked, because it doesn’t fit with the conventional understanding of cancer development. A paper from last month¹ proposes a new and rather troubling model.

People rarely die of a primary tumor; the direct cause of death is usually metastatic cancer. The prevailing models of cancer development suggest that metastasis is a late event in cancer development. (I believe this concept was first articulated by Fidler in 1978,² although in retrospect this paper only argues that metastasis is the product of a subset of selected cells from the tumor — the concept that a tumor is not a homogenous entity — not that the selection process that leads to the metastatic subset must necessarily occur late in the cancer’s progression.) Hanahan and Weinberg, in their immensely influential 2000 paper “The Hallmarks of Cancer”,³ discussed “Tissue Invasion and Metastasis” as the sixth and final “acquired capabilities” of a tumor, and show metastasis as the final event in four of the five pathways they illustrated, and as the penultimate event in the other one (see the figure to the right here; click for a larger version).

However, there’s also some evidence that metastasis isn’t necessarily a very late event in cancer development. For example, Engel et al. worked out the point at which metastases from breast cancers must have occurred, based on their growth rates, and determined that metastases must often happen quite early in the course of the tumor: ⁴

Our hypothesis of an early MET [metastasis. IY] initiation questions the morphological and genetic correlation between the primary tumour and MET, because the primary tumour at the time of dissemination would have been much smaller and probably had prognostically favourable characteristics.

The paper I’m talking about here,¹ from Hüsemann et al, takes this argument a couple of steps further. Not only can metastases arise very early in the course of cancer development, they say, metastases can even arise before the cells are fully malignant. Using a mouse mammary carcinoma model in which mammary tumors arise in a predictable fashion, they were able to detect the premalignant mammary cells in the bone marrow as early as 4 weeks - before outright tumors had arisen in the mammary tissue itself — and these early-spreading cells were able to cause cancer in irradiated (immune-deficient) mice after bone marrow transplant.

One provocative finding of our study is that, in mouse models of breast cancer, large tumors seed neither more nor genetically further-advanced cancer cells than do small lesions… Thus, the ability of metastatic dissemination does not appear to be the result of selection of tumor cells within the tumor. Rather, the data suggest that tumor cells disseminate early and will be selected for outgrowth at distant sites.

(My emphasis.)

If metastases spread very early, then why do we even see “primary tumors”? Why don’t the metastases arise at the same time as the “primaries”, or even earlier — so that all we see is a metastatic cancer with no primary tumor? (In fact, this is exactly what is seen in a lot of tumors. Some 5 - 10% of tumors present as metastases with an unknown primary tumor. ⁵ However, the question still applies to the remaining 90-95% of tumors.)

The most likely explanation is that there is some growth restriction on metastases. For example, perhaps the primary tumor secretes growth factors systemically, which permits the metastases to expand. Or — and this is a possibility the authors didn’t really discuss, so I’m not sure if there’s some obvious flaw that I’m missing — the immune system may hold the small tumors in check for a while until the metastases manage to evade equilibrium with the immune system. I wonder if this could be a regulatory T cell (TReg) effect, in which the primary tumor is able to expand and then conditions infiltrating lymphocytes to become TRegs; we know that TRegs circulate, so perhaps these circulating TRegs then shut down the ongoing response to the metastases as well, and allow them to grow out.

In any case, this observation (assuming it holds up) changes some of the concepts of cancer therapy, I think. If the metastases are already out there, then some of the rationale for removing the primary tumor is gone. On the other hand, there’s a potential opportunity there, too: If something prevents metastases from growing out and causing a problem for a long time, perhaps that something can be harnessed and used to prevent outgrowth for a lifetime.

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1. Yves Hüsemann, Jochen B. Geigl, Falk Schubert, Piero Musiani, Manfred Meyer, Elke Burghart, Guido Forni, Roland Eils, Tanja Fehm, Gert Riethmüller and Christoph A. Klein. (2008). Systemic Spread Is an Early Step in Breast Cancer. Cancer Cell, 13(1), 58-68. DOI: 10.1016/j.ccr.2007.12.003[↩][↩]
2. Fidler, I. J. (1978). Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 38, 2651-2660.[↩]
3. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.[↩]
4. Engel, J., Eckel, R., Kerr, J., Schmidt, M., Furstenberger, G., Richter, R., Sauer, H., Senn, H. J., and Holzel, D. (2003). The process of metastasisation for breast cancer. Eur J Cancer 39, 1794-1806.[↩]
5. van de Wouw AJ, Janssen-Heijnen MLG, Coebergh JWW, Hillen HFP (2002) Epidemiology of unknown primary tumours; incidence and population-based survival of 1285 patients in Southeast Netherlands, 1984-1992. European Journal of Cancer 38.:409-413 [↩]

A while ago, talking about tumor development, I said that “tumors normally develop ways to avoid immunity.” It’s that ability to evade the immune system that allows tumors to escape from the equilibrium phase, when they’re a mere harmless handful of cells held in check by the immune system, and become outright clinically-detectable tumors.

How do tumors avoid immunity?

There are many ways. Remember that tumors are not like viruses; each tumor arises de novo and has only its host’s lifespan in which to evolve. It has no connection to other tumors of the same kind; there is no evolutionary linkage. All human cytomegaloviruses have a common ancestor, but no two colon cancers have the same ancestor. ¹ That means that each tumor must find its own solution to every problem rather than relying on its ancestors’ solutions. (And if most colon carcinomas, say, adopt the same solution, we have to look at what factors make that solution particularly accessible to, or appropriate for, that particular type of cancer.)

That said, there are some mechanisms that are very widely used by tumors to evade the immune system, and regulatory T cells (TRegs) are one of them. That’s probably at least in part because TRegs’ natural functions include suppressing immune responses to self antigens and reducing inflammation in chronic exposure to antigen. It’s relatively easy to get the TRegs to do their job a little more enthusiastically.

(TRegs are T cells that specifically down-regulate immune responses; without TRegs, immune responses explode and cause massive damage. People without TRegs have terrible, usually fatal, autoimmune and inflammatory disease, so you don’t want to just blithely eliminate them to get a “better” immune response. )

It was suggested nearly 30 years ago that TRegs contributed to tumor growth,² but the whole I-J fiasco set the field back a long way, and it wasn’t until relatively recently that the questions were revisited. ³ It’s now pretty clear that, in fact, TRegs are often abundant in tumors, and actively shut down immune responses to the tumors. For example, it’s been shown recently that tumors with relatively more TRegs have a worse prognosis:⁴

In patients with undesirable outcome, the balance is tipped in favor of Tregs (high Tregs and low activated CTLs), whereas in patients with relatively desirable outcome, the balance is tipped toward effector T cells (low Tregs and high activated CTLs).

Eliminating TRegs from mice with experimental tumors caused rejection of the tumors,⁵ so as you’d expect, there’s a lot of interest in this sort of approach to cancer therapy.

Relevant previous postsCancer:
• … and immune escape
• … and TLRs
• … and immunity
• … the Three E’s of
• … and equilibrium
Regulatory T cells:
• … and the I-J story
• … and persistent viruses
A small step forward was described recently in PNAS. ⁶ “Classic” TRegs are antigen-specific; they recognize antigen just as do T Helper cells, but instead of responding by stimulating immune responses, the TRegs respond to their antigen by suppressing local responses. There are also non-antigen-specific cells that act something like TRegs (though not as effectively). This paper shows that patients with melanoma have circulating (not just tumor-infiltrating) TRegs that are specific for tumor antigens.

 

This is a nice little paper, but I don’t think it represents a very large or surprising advance.⁷ It’s already known that tumor-infiltrating TRegs can be tumor-antigen-specific;⁸ it’s known that patients with melanoma have increased numbers of circulating TRegs;⁹ and it’s also been shown that tumor-specific T cells are in circulation in patients with melanoma. ¹⁰ So this (as far as I can see) extends several prior observations, but in an unsurprising direction.

Also, unless I’m missing something (which wouldn’t be new), I don’t see just how knowing or identifying circulating TReg antigens will help with treatment or prognosis. One possibility that occurs to me is that these circulating TRegs may help protect metastases from rejection. If the TRegs were all infiltrating the tumor, then perhaps the immune system would be able to deal with the small number of metastatic cells that are spreading throughout lymphatics and so on, whereas if TRegs are also circulating then the metastatic cells might be protected. I’m not entirely sure about the actual relevance of this, because even if TRegs were only tumor-infiltrating I think there would be plenty of opportunity to tolerize tumor-specific T cells.

Still, it’s always important to understand the system.

Just as a final cautionary note: As I said up at the top, tumors are all different. Different classes of tumors may be particularly able to move along certain immune evasion pathways because of their underlying characteristics. For example, inflammatory infiltrate within some tumors is a good sign¹¹ (presumably because it implies that there is an active immune response against the tumor), yet in other classes of tumor the same kind of infiltrate is a bad sign, perhaps because it indicates TReg infiltration and a suppressive environment. Even within classes of tumors there are inevitably variations. It may not be possible to develop universal rules for tumor immune treatment; but it may be possible to find some useful guidelines.

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1. With bizarre exceptions like transmissible canine veneral tumor and Tasmanian Devil tumor. [↩]
2. Berendt, M.J. & North, R.J., 1980. T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. The Journal of experimental medicine, 151(1), p.69-80.[↩]
3. A nice review: Zou, W., 2006. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol, 6(4), p.295-307. [↩]
4. Gao, Q. et al., 2007. Intratumoral Balance of Regulatory and Cytotoxic T Cells Is Associated With Prognosis of Hepatocellular Carcinoma After Resection. J Clin Oncol, 25(18), p.2586-2593. DOI: 10.1200/JCO.2006.09.4565 [↩]
5. Shimizu, J., Yamazaki, S., & Sakaguchi, S., 1999. Induction of Tumor Immunity by Removing CD25+CD4+ T Cells: A Common Basis Between Tumor Immunity and Autoimmunity. J Immunol, 163(10), p.5211-5218.
   and
   Viehl, C. T., Moore, T. T., Liyanage, U. K., Frey, D. M., Ehlers, J. P., Eberlein, T. J., Goedegebuure, P. S., and Linehan, D. C. (2006). Depletion of CD4+CD25+ regulatory T cells promotes a tumor-specific immune response in pancreas cancer-bearing mice. Ann Surg Oncol 13, 1252-1258.[↩]
6. Vence, L. et al., 2007. Circulating tumor antigen-specific regulatory T cells in patients with metastatic melanoma. Proceedings of the National Academy of Sciences, 104(52), p.20884-20889. [↩]
7. I mention it here mainly because it reminded me that this is a subject I wanted to talk about.[↩]
8. see Wang, R., 2006. Functional control of regulatory T cells and cancer immunotherapy. Seminars in Cancer Biology, 16(2), p.106-114. for a review[↩]
9. McCarter, M.D. et al., 2007. Immunosuppressive dendritic and regulatory T cells are upregulated in melanoma patients. Annals of surgical oncology, 14(10), p.2854-60.[↩]
10. For example, Michalek, J. et al., 2007. Detection and Long-Term In Vivo Monitoring of Individual Tumor-Specific T Cell Clones in Patients with Metastatic Melanoma. J Immunol, 178(11), p.6789-6795.

    and Bioley, G. et al., 2006. Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers. Journal of immunology 177(10), p.6769-79.[↩]

11. For example, Eerola AK, Soini Y, Paakko P. A high number of tumor-infiltrating lymphocytes are associated with a small tumor size, low tumor stage, and a favorable prognosis in operated small cell lung carcinoma. Clin Cancer Res. 2000; 6: 1875-1881[↩]

Oncolytic VSV (gold) infecting lung tumors1

Oncolytic viruses are a concept I’d like to be more excited by than I am.² It’s an idea that seemed really exciting when I first came across it, but the more I thought about it the more dubious I was. But a recent paper helps me feel better about at least two of my worries.

The concept is a straightforward one. Viruses are good at killing cells.³ Why not have them infect cells that we want to die? That would be, for example, cancer cells. So all you need to do is find or make a virus that only grows in cancer cells, and you’re cured. Simple! Tomorrow we’ll fix global warming!

There’s the obvious problem with this: How can you find (or make) a cancer-specific virus? In principle the answer is the same as with chemotherapy; you use the ways cancer cells are different from normal as targets. This isn’t as hard as you might think. Lots of the things that make cancer cells cancerous are similar to the things viruses like. Viruses often drive infected cells into a cancer-like state that is more hospitable to the virus — friendly to nucleic acid replication, replication, unresponsive to death signals, independent of the signals that normally regulate growth. So lots of viruses are already kind of pre-adapted to replicate well in cells with a cancerous phenotype, and it doesn’t take all that much tweaking to make them adapted to only replicate well in cancer cells.

(After writing this post, it occurred to me that this is actually topical! I don’t usually do the topical blog post thing, but the background in “I Am Legend” has an anti-cancer virus, isn’t it? I haven’t seen it myself, with the grant-writing and the teaching and the two little kids,⁴ but have I actually tied a current entertainment topic into “Mystery Rays”? Fame and fortune is certain to come my way!)

Oncolysis through the ages

The first runs at this technique that I knew of⁵ used mutant herpesviruses,⁶ but I think that much of the buzz came from work with defective adenoviruxes, especially the ONYX-015 virus.⁷ The approach here was based on the observation that adenoviruses (like many other viruses) normally inactivate p53 during infection. p53 is a multifunctional growth regulator that is very often also inactivated in cancers, for the same reason as viruses like to inactivate it:it oten triggers death in cells with unchecked growth. Adenoviruses lacking the gene that inactivates p53 (their E1B gene) can only efficiently infect cells lacking p53 — which would usually be, of course, cancer cells.

As well as herpesviruses and adenoviruses, though, all sorts of other viruses have been used.⁸ One interesting approach is vesicular stomatitis virus. This is a very, very innocuous virus in normal people, partly because VSV is extremely sensitive to interferon. (VSV is used in bioassays for interferon release, because even tiny amounts of interferon completely block the virus’s replication.) So which kind of cells often aren’t responsive to interferon? Right; cancer cells, as part of their own immune evasion pathway, frequently disable their interferon responses. VSV doesn’t infect normal cells, but does infect, and kill, tumor cells.⁹

Questions and (maybe) answers

Anyway, the first question, of specificity, is more or less under control.¹⁰ Three questions that had made me rather dubious about the concept, though, still remained:

1. Getting the virus to the tumor …
2. Especially in the face of an immune response.
3. Killing all of the cancer cells, not a mere 99% of them (from which the cancer will rapidly recover).

Malignant melanoma cells in lymph node

A paper in Nature Medicine¹¹ offers encouragement on all of those.

They used VSV as their cancer killer, and their twist here was to deliver it by loading it onto T cells. T cells naturally traffic to lymph nodes, and quite a few tumors metastasize through lymph nodes; the T cell therefore acts as a ferry to deliver its deadly viral cargo to the metastasizing tumor. (The goal here was not to clear the primary tumor, but to prevent metastases, which are often the major problem.  However, they did see some effect on the primary tumor, too, in some cases.) When it reaches the lymphoid tissue, it delivers the passenger virus to the cancer cells, the only ones that the VSV can productively infect (since the cancer cells are the only ones that have mutated their interferon pathway). This is an interesting idea, though limited in this form — I wonder about using antigen-specific T cells instead, to target the virus to a specific site — and it seemed to work quite well.

The two more interesting points to me were kind of peripheral to their main point. First, they find that once the virus killed some cancer cells, there was anti-tumor protection even after the virus was all cleared, and this was probably because of the immune response,¹² which was triggered by the cell death initially caused by the virus:

In vivo tumor cell purging resulted both from direct viral oncolysis by virus released from the T cell carriers and from the priming of protective antitumor immunity, which prevents repopulation by further waves of cells metastasizing from the primary tumor.

– just as described in the paper by Apetoh et al¹³ that I talked about here. The authors suggest that because the cancer metastases are being killed in the lymph nodes, rather than in the bulk of the tumor (which is generally a highly immunosuppressive environment) the immune response was more efficient. That starts to get past my concern #3 above, because it offers multiple attacks on the tumor, not just the virus.

The other point is that the virus could reach the cancer reasonably well even in the face of an anti-viral immune response; the trick was to use just enough virus to kill the tumor cells, without getting enough on the T cells to trigger an immune response:

In virus-immune mice, T cells loaded with large amounts of VSV (MOI 1 or 10) could not keep DLNs or spleens free of tumors. However, T cells loaded with fewer viruses (MOI 0.1) still protected even virus-immune animals from tumor colonization of the DLN and spleen

The data are still very preliminary and inconclusive, but certainly it’s a step in the right direction, and I feel better about this whole approach than I did before reading the paper.

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1. Carrier Cell-based Delivery of an Oncolytic Virus Circumvents Antiviral Immunity. Anthony T Power, Jiahu Wang, Theresa J Falls, Jennifer M Paterson, Kelley A Parato, Brian D Lichty, David F Stojdl, Peter A J Forsyth, Harry Atkins and John C Bell. Molecular Therapy (2007) 15, 123-130. [↩]
2. That sentence needs a road map, but you got here eventually, didn’t you.[↩]
3. At least, lytic viruses are.[↩]
4. And the running and the screaming and the monkeys in the hair[↩]
5. I realize now, though, that the concept arose long before that, apparently in the 1950s. For example: Love R, Sharpless GR. Studies on a transplantable chicken tumor, RPL-12 lymphoma. II. Mechanism of regression following infection with an oncolytic virus. Cancer Res. 1954 Oct;14(9):640-7.http://dx.doi.org/10.1126/science.1851332 though I don’t know much about those studies other than the titles [↩]
6. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Science. 1991 May 10;252(5007):854-6. [↩]
7. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. Nat Med. 1997 Jun;3(6):639-45.
   and
   An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A, McCormick F. Science. 1996 Oct 18;274(5286):373-6. [↩]
8. And I have no idea which, if any, is the mo