Mystery Rays from Outer Space

We know that viruses cause a significant minority of human cancers, but we don’t know quite how many, or which, cancers are viral. It’s not as easy as you might think to tell.

The link between viruses and cancer was one of the major breakthroughs in cancer biology, but you could also make a case that that link set cancer research back several years. (Cancer viruses were shown, in chickens, in 1911¹, but it wasn’t until the 1960s that interest in the concept took off, with Epstein’s demonstration of Epstein-Barr virus (EBV)² in some human tumors. ) Studying viral oncogenesis has led to huge advances in our understanding of the fundamental biology of cancers. The problem was that there was a general assumption, in the 1960s and 1970s, that viruses were directly responsible for the vast majority of human tumors. If so, all we needed to do was to identify the viruses, develop vaccines against them, and voila! No more cancer!

Of course, it wasn’t that easy. For all the fundamental advances from this concept, it’s been relatively unproductive as far as the bedside is concerned. Most human tumors are not caused by viruses,³ and even those that are, have been really resistant to treatment via direct anti-viral approaches.⁴ The research units that were established in the 1970s to look at the virus/tumor connection have mostly either disbanded, or taken a different direction now.

Mouse polyomavirus

In spite of that, there’s still new and exciting stuff coming out. Most recently,⁵ Yuan Chang and Patrick Moore identified a new cancer-causing virus of humans. (This was their second breakthrough virus; in 1994⁶ they also found the second-most recent cancer-causing virus of humans, Kaposi’s Sarcoma Herpesvirus.)) This brings to six the number of clearly-linked human cancer viruses: papillomaviruses, HTLV-1, hepatitis B virus, EBV, Kaposi’s Sarcoma Herpesvirus, and the new one, Merkel cell polyomavirus.

I keep meaning to talk about the discovery of Merkel cell polyomavirus, but I’ll set that aside for now. Very briefly, last fall Chang and Moore showed that the presence of the virus is linked to a fairly rare tumor, Merkel cell carcinoma, and they and others have now confirmed the epidemiological association and demonstrated a mechanism for causing tumors. We’re at the stage now of trying to understand the normal biology of the virus. As with most (and all human) cancer viruses, the virus is much more widespread than the tumor. How widespread it is? How does it spread? Does it cause other disease — in particular, is it involved in other tumors?

That last is a particularly interesting question, because although Merkel cell tumors are rare, there are a number of common tumors that could, conceivably, also be caused by the virus; and if the virus causes even a subset of those, then it could be a common cause of cancer rather than an unusual one. So far, though, I think the evidence suggests that the virus is fairly limited in the damage it causes; quite a few groups have looked for the virus in other types of cancer, and although it may be occasionally found⁷, that most likely reflects general background infection with the virus rather than a causative role.

One exception is a recent paper⁸ which suggested that the virus might be associated with a subset of squamous cell carcinomas. SCC are a pretty common form of skin tumor, so if MCPyV is a cause of even a subset of SCC it might be a significant cause of cancer.

The problem is that the other studies on MCPyV have shown that it’s out there even in normal, non-diseased humans⁹ — as you’d expect; the virus must be able to spread and circulate within the human population somehow, and the version of the virus found in Merkel cell tumors is damaged and likely can’t spread, so there must be virus replicating in normal tissues. In this paper, the authors find the virus in a subset of SCC cancers, but I would like to know how many they’d find in normal human skin using the same techniques –  the 15% of positive tumor samples may or may not be significantly different. On the other hand, the SCC-associated virus did show a similar molecular signature to that found in Merkel cell cancer,¹⁰ suggesting a causative role. Right now, I’m not completely convinced, but am definitely intrigued.

One really interesting point to add is that — if MCPyV really does cause a significant number of tumors — then it’s been missed all these years, despite high interest in searching for human cancer viruses, until new techniques were applied to the right samples in the right way. Are there other human cancer viruses out there, waiting for the right technique? Is it still possible that most human cancers are caused by viruses? I think that’s pretty unlikely, but the door is still open a crack.

1. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. Rous, P. 1911. J. Exp. Med. 13:397–411.[↩]
2. Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. MA Epstein, BG Achong and YM Barr. Lancet 1 (1964), pp. 702–703.[↩]
3. The usual educated guess is 20%[↩]
4. One major exception, of course, being papilloma virus vaccines, which are a direct outcome of this line of research. But it’s taken a long time to come to fruition. You could also point to vaccination against hepatitis B virus, and a number of veterinary vaccines, as clinical advances arising from the virus/cancer research. But I think it’s fair to say that the energy put into this has been fundamentally, but not clinically, well spent.[↩]
5. Feng, H., Shuda, M., Chang, Y., & Moore, P. (2008). Clonal Integration of a Polyomavirus in Human Merkel Cell Carcinoma Science, 319 (5866), 1096-1100 DOI: 10.1126/science.1152586[↩]
6. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma.
   Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS.
   Science. 1994 Dec 16;266(5192):1865-9.[↩]
7. For example, Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma, lymphoid tissues and lymphoid tumors.
   Shuda M, Arora R, Kwun HJ, Feng H, Sarid R, Fernández-Figueras MT, Tolstov Y, Gjoerup O, Mansukhani MM, Swerdlow SH, Chaudhary PM, Kirkwood JM, Nalesnik MA, Kant JA, Weiss LM, Moore PS, Chang Y.
   Int J Cancer. 2009 Sep 15;125(6):1243-9[↩]
8. Dworkin, A., Tseng, S., Allain, D., Iwenofu, O., Peters, S., & Toland, A. (2009). Merkel Cell Polyomavirus in Cutaneous Squamous Cell Carcinoma of Immunocompetent Individuals Journal of Investigative Dermatology DOI: 10.1038/jid.2009.183[↩]
9. Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays.
   Tolstov YL, Pastrana DV, Feng H, Becker JC, Jenkins FJ, Moschos S, Chang Y, Buck CB, Moore PS.
   Int J Cancer. 2009 Sep 15;125(6):1250-6[↩]
10. Shuda, M., Feng, H., Kwun, H., Rosen, S., Gjoerup, O., Moore, P., & Chang, Y. (2008). T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus Proceedings of the National Academy of Sciences, 105 (42), 16272-16277 DOI: 10.1073/pnas.0806526105 [↩]

Tammer Wallaby

Everyone knows that the MHC is in the MHC, right? Well, it’s not necessarily so.

That’s not as tautological as it sounds. MHC (major histocompatibility complex) can refer to either the protein complex, or to the genomic region. In most species the genes encoding MHC proteins are clustered together into a distinct region of the genome that usually contains a bunch of genes that are functionally, and in some cases structurally, linked. For example, the human MHC genomic region contains not only many MHC class I genes, but also the TAP genes that are required for their function (outline of function here); MHC class II genes, and a number of genes required for their function; some proteasome subunits that are also involved in the antiviral MHC function; and so on. There’s a crude map here, and a somewhat more detailed one here; in humans and many other species the MHC genomic region is densely packed with genes, many of which are immunologically important.

Although the details are different the same concept applies to many vertebrates — chickens ¹, Xenopus (frogs),² and sharks,³ for example, have only one classical MHC class I gene, but it’s recognizably in the MHC genomic region, tightly linked to TAP and not quite as tightly linked to MHC class II genes.

Why are these functionally-related genes clustered together? There are probably a bunch of reasons, and the reasons may actually be different for different species. A recent paper,⁴ showing a partial exception to the rule, makes some interesting suggestions. Siddle et al⁵ have looked at wallaby MHC genes and find that their classical MHC class I genes are actually scattered throughout the genome, and are not in the MHC region.

First of all, what’s the advantage of having a single MHC class I gene tightly linked to TAP (probably the primordial organization)? TAP transports peptides to the MHC (again, cartoons of MHC class I function here), and the MHC then presents the peptides to T cells for antiviral surveillance. That means that TAP needs to handle the same kinds⁶ of peptides that the MHC class I does. By linking the genes for TAP and the MHC class I, the two can evolve in tandem — if a particular MHC class I gene likes to bind peptides ending with, say, arginine, then it can co-evolve along with a TAP that likes to transport peptides ending in arg. MHC class I genes are extremely variable, and in non-mammalian species, TAP genes are also relatively variable, ⁷ arguing for this kind of co-evolution.

The problem with this organization is that it only really allows one MHC class I specificity. If the TAP has a certain, strong, specificity (ending with Arg, say), and you had several different MHC class I proteins each with different peptide preferences (one that wants peptides ending with Arg, but another that wants peptides ending in tyrosine), then some of them wouldn’t match the TAP peptides and would go wanting.

(By the way, this makes a start at explaining a paper that puzzled me some time ago [post is here]. The chicken B21 MHC class I allele was said to have very weak peptide preferences — allowing “promiscuous peptide binding”. But if TAP has strong peptide preferences, then the MHC is only going to bind to a limited subset of peptides, no matter how promiscuous the MHC is itself. That doesn’t explain everything, but it makes a start. I should mention, though, that a different group looking at B21 did, in fact, identify peptide binding preferences,⁸ suggesting that binding isn’t actually promiscuous; but now I wonder if they were detecting TAP preferences rather than MHC.)

Although it’s not a hundred percent clear why MHC class I is so diverse,⁹ according to the most plausible explanations the advantages of diversity are going to be increased if you have several different MHC class I genes, with different peptide-binding properties. If you can hoick the MHC away from TAP, then, you’d allow the MHC to start diversifying independent of TAP. You’d probably need TAP to now be fairly peptide-promiscuous (which it is, in most mammals), and shift the peptide specificity over to the MHC class I molecules themselves.

Humans, mice, and most mammals that have been looked at do this (separate MHC class I from TAP, and have multiple MHC class I alleles) by sliding the MHC class I genes over to the side, remaining within the MHC genomic region but becoming far enough separated from TAP that the genes can evolve more or less independently. Wallabies apparently have done the same thing functionally, but instead of sliding over and keeping the genes in the MHC genomic region, they’re scattered throughout the rest of the genome, apparently via retrotranspon-mediated transposition.

The classical class I have moved away from antigen processing genes in eutherian mammals and the wallaby independently, but both lineages appear to have benefited from this loss of linkage by increasing the number of classical genes, perhaps enabling response to a wider range of pathogens.⁴

Incidentally, it occurs to me that there is an extra cost to this increased diversity. By un-linking TAP specificity from MHC class I peptide preferences, mammals force TAP to be highly promiscuous, and to transport a wide range of peptides — unlike in chickens, TAP no longer “knows” what MHC allele it’s dealing with and has to offer every possible peptide sequence that any of the thousands of MHC class I alleles could bind. That means that there must be a vast amount of wasted peptides transported into the endoplasmic reticulum; in contrast, I would expect chickens, for example, to predominately only transport peptides that can bind to their MHC class I. If you suspect, as I do, that peptides are intrinsically toxic at high doses, then mammals must have developed (or enhanced) some mechanisms for destroying the extra peptides, that non-mammalian vertebrates don’t have to worry about. I have a guess as to what the mechanism might be, but I’m not sure exactly how to test it right now.

1. Kaufman J, Milne S, Gobel TW, Walker BA, Jacob JP, Auffray C, Zoorob R, Beck S: The chicken B locus is a minimal essential major histocompatibility complex.Nature 1999, 401:923-925. [↩]
2. Nonaka M, Namikawa C, Kato Y, Sasaki M, Salter-Cid L, Flajnik MF: Major histocompatibility complex gene mapping in the amphibian Xenopus implies a primordial organization.Proc Natl Acad Sci U S A 1997, 94:5789-5791. [↩]
3. Ohta, Y., McKinney, E.C., Criscitiello, M.F., and Flajnik, M.F. 2002. Proteasome, TAP, and class I genes in the nurse shark Ginglymostoma cirratum: Evidence for a stable class I region and MHC haplotype lineages. J. Immunol. 168:771-781.[↩]
4. Siddle, H., Deakin, J., Coggill, P., Hart, E., Cheng, Y., Wong, E., Harrow, J., Beck, S., & Belov, K. (2009). MHC-linked and un-linked class I genes in the wallaby BMC Genomics, 10 (1) DOI: 10.1186/1471-2164-10-310[↩][↩]
5. The same Siddle, I believe, who I recently cited for work on the Tasmanian Devil genome a couple of times[↩]
6. That is, the same general amino acid sequences[↩]
7. Ohta Y, Powis SJ, Lohr RL, Nonaka M, Pasquier LD, Flajnik MF. Two highly divergent ancient allelic lineages of the transporter associated with antigen processing (TAP) gene in Xenopus: further evidence for co-evolution among MHC class I region genes. Eur J Immunol. 2003 Nov;33(11):3017-27.[↩]
8. Sherman MA, Goto RM, Moore RE, Hunt HD, Lee TD, Miller MM (2008) Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-B haplotypes associated with opposite responses to Marek’s disease. Immunogenetics 60:527–541.[↩]
9. MHC diversity: see here and its linked articles, also here and here and linked articles therein[↩]

A year or so ago, the first time I mentioned transmissible tumors, I dismissed Tasmanian Devil Facial Tumor disease as not particularly surprising. As I said more recently, I’ve changed my mind about that, almost entirely because of a chat with Elizabeth Murchison at the Origins of Cancer symposium last month, who told me a couple of things I hadn’t known about the situation.

TDFT is a transmissible tumor, a cancer that arose in one Tasmanian Devil some time ago (at least a decade ago, and maybe much longer) and that is now spreading throughout the Tasmanian Devil population — killing a vast number of them. It’s a tumor that’s spread when these highly territorial animals¹ bite each other on the face; the tumor then grows on the bitten animal’s face and eventually makes it unable to eat properly. Nearly 90% of Tasmanian Devils, in affected areas, have died. This is a catastrophe.

(The good news, such as it is, may be that the Devils as a population are adapting to the tumor to some extent — not by resisting the tumor, though, but through rapid selection for much earlier breeding. ² By reproducing earlier, the population may be maintained even though the Devils are dying as adults. Of course, the population that “survives” won’t be the same as those the existed earlier — they’ll be younger, they’ll have all kinds of different characteristics because they’re no longer being selected for adult survival — but at least it might open a window to save the species.)

Cancers are not, in general, transmissible.  Each new cancer is a new event, that arises from normal cells of the affected individual.  A transmissible cancer would be like an organ transplant — it would be rapidly rejected, because individuals in a population have different MHC molecules, and the immune system rapidly and aggressively rejects cells with the wrong MHC. So there are two possible reasons why a tumor could, in fact, become transmissible. Either the individuals in a population do not have different MHC molecules on their cells, or the tumor can transmit between individual in spite of different MHC molecules.

The explanation for the spread of TDFT seemed to be the former: It was claimed that Tasmanian Devils have very little MHC diversity, so that they can’t reject the tumors³. Even though MHC is normally highly diverse in a population, there are certainly populations — especially small, island, threatened populations, like Tasmanian Devils — that have very limited MHC diversity, so this seemed reasonably plausible. ⁴ This would be a fairly well-understood mechanism of tumor spread, which is why I said it wasn’t that interesting.

The other possibility is that the tumor could spread between individuals in spite of them having different MHC molecules. This is not supposed to happen, according to our understanding of MHC and organ transplants; but in fact, it’s the way the only other extant transmissible tumor (Canine Transmissible Venereal Tumor) spreads.  We have no idea why that happens, which makes it interesting. (A number of ways have been proposed by which CTVT avoids rejection. I won’t go into them in any detail here: none of them, as far as I can see, are unique to CTVT, but rather are very common molecular changes in tumors of all kinds, and yet other tumors are not transmissible; so I don’t consider any of these suggestions to explain the unique feature of CTVT.)

Anyway, as I say, the explanation for TDFT was simple enough: The tumor spread because Tasmanian Devils aren’t genetically diverse. But there are three major arguments, I’ve learned, that say that explanation is wrong.

First: Tasmanian Devils are not, in fact, spectacularly genetically homogenous. ⁵ They’re not as diverse as you’d like to see, but they’re not completely homogenous. In particular, there are two clear, genetically distinct, populations of Devils in Tasmania, one in the East, and another in the Northwest.

Second: Murchison told me that Tasmanian Devils — even those in the same sub-population — vigorously reject each others’ skin grafts. This is what’s supposed to happen with skin grafts, of course. It implies that the Devils do not, in fact, have the same MHC; and in my opinion it’s a much stronger experiment than those in the original homogenous-MHC paper. ³ If Devils reject skin grafts from each other, then they ought to reject tumors from each other — in other words, even if the tumor can take in one individual, then it should be rejected in another, so the tumor should not spread throughout the population. The skin graft finding hasn’t, as far as I know, been published, but if it holds up, it’s a strong argument against homogenous MHC.

Third: Murchison also told me that the tumor is spreading into Devils in the Northwest. ⁶ As I said, the Northwestern and Eastern Tasmanian Devils are clearly distinct populations, with different genetic characteristics. ⁷  If the tumor can spread between them, then the tumor isn’t relying on genetic homogeneity for its transmission.

So the evidence for MHC homogeneity is not good, the evidence that the tumor requires MHC homogeneity is not good, and the only precedent we know of, CTVT, does not require MHC homogeneity for its transmission.

It seems that the two transmissible tumors we know of may be much more similar than I had thought.

1. Incidentally, when I was looking for images of Tasmanian Devils, I found it interesting that the great majority of images I found using Google Image Search showed an open-mouthed, angry Devil, about to take a chomp out of something.  I was going to comment on that, and then I went to Flickr and did the same search, finding instead thousands of pictures of peaceful, timid, close-mouthed Devils.  I think the popular images, put by Google at the top of the list, as so popular because they reinforce the Devil stereotype, and the Flickr photos are a more accurate reflection of their true nature.[↩]
2. Jones, M., Cockburn, A., Hamede, R., Hawkins, C., Hesterman, H., Lachish, S., Mann, D., McCallum, H., & Pemberton, D. (2008). Life-history change in disease-ravaged Tasmanian devil populations Proceedings of the National Academy of Sciences, 105 (29), 10023-10027 DOI: 10.1073/pnas.0711236105[↩]
3. Siddle, H. V., Kreiss, A., Eldridge, M. D., Noonan, E., Clarke, C. J., Pyecroft, S., Woods, G. M., and Belov, K. (2007). Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial. Proc Natl Acad Sci U S A 104:16221-16226[↩][↩]
4. Olivia Judson had a very good summary of the argument in her blog; she also made the important point that MHC diversity, or lack of it, is clearly not the only factor involved in the tumor spread.[↩]
5. JONES, M., PAETKAU, D., GEFFEN, E., & MORITZ, C. (2004). Genetic diversity and population structure of Tasmanian devils, the largest marsupial carnivore Molecular Ecology, 13 (8), 2197-2209 DOI: 10.1111/j.1365-294X.2004.02239.x[↩]
6. Again, as far as I know this isn’t published yet.[↩]
7. The authors of the 2004 paper didn’t specifically look at MHC diversity, though; so it remains possible, if unlikely, that the MHC is relatively homogenous while the rest of the genome is diverse.  This is completely the opposite of the usual situation, so I think it’s not likely.[↩]

As the overwhelming majority of the mutations in cancer cells are unrelated to malignancy, the mutation-generated epitopes shall be specific for each individual tumor, and constitute the antigenic fingerprint of each tumor. These calculations highlight the benefits for personalization of immunotherapy of human cancer, and in view of the substantial pre-existing antigenic repertoire of tumors, emphasize the enormous potential of therapies that modulate the anti-cancer immune response by liberating it from inhibitory influences.

–Srivastava N, Srivastava PK (2009)
Modeling the Repertoire of True Tumor-Specific MHC I Epitopes in a Human Tumor.
PLoS ONE 4(7): e6094. doi:10.1371/journal.pone.0006094

(My emphasis)

(See many of my previous posts here for more information)

Dogs & Wolf (Gotthilf Heinrich von Schubert, 1872) Naturgeschichte der Säugethiere: mit colorirten Abbildungen zum Anschauugs-Unterricht für die Jugend. (Esslingen : Schreiber, 1872)

Cancer is a creepy disease. Your own cells turn on you, mindlessly and blindly destroying themselves — because the only way a cancer can survive is for its host to survive; unlike viruses, cancers don’t spread from their original carrier to new hosts. Each cancer is a new and unique event, and each cancer is a terminal event, with no future, no children, no transmission. ¹ Cancers don’t spread between individuals, because they would essentially be tissue transplants; if you try to transplant skin (or whatever) between two animals, the skin will be rapidly rejected because of mismatched MHC alleles, and the same is true for tumors.

In theory, tumors could spread from one identical twin to another (I don’t know of cases where this has occurred, though). Tumors might also theoretically spread within a population that has little MHC diversity. Cheetahs, for example, notoriously don’t have much MHC diversity,² probably due to a genetic bottleneck some 10,000 years ago,³ and tolerate random skin grafts very well;⁴ it’s conceivable that a tumor that arose in one cheetah could spread throughout the population. But MHC is generally diverse, and becomes diverse very rapidly, so these sort of low-diversity populations are unusual (though they may be more common than generally realized). And so, as I say, tumors are dead ends and don’t spread from their original host.

But there are always exceptions, and we know of a couple exceptions for this principle as well. Tasmanian Devil Facial Tumor is one; canine transmissible venereal tumor (CTVT) is the other. The last time I talked about this, I said that there wasn’t anything particularly surprising about TDFT. I’ve changed my mind about that, for reasons I’ll talk about later. Here I want to give a quick update on CTVT, which is still even more interesting than TDFT.

CTVT is just what it sounds like: an infectious tumor of dogs. It can be transmitted from one dog to the next, mainly during sex. There’s no sign of a viral agent, and in fact the tumor cells are distinct from the host — that is, the tumor cells are not host cells that have been transformed by, say, a virus, but are foreign agents altogether, cells from an ancient animal that have been passed along in this way, generation after generation, spreading throughout the world. Cells from a CTVT on a German Shepherd in Italy are virtually identical to those from a CTVT on a poodle in Brazil, but the tumor’s cells are very different from their hosts’: “… the differences in genome content between dog and CTVT are substantially greater than those between different CTVT samples.“ ⁵

The last time I talked about this I cited a paper⁶ that showed that the original host of CTVT was actually very ancient: “Our analysis of divergence of microsatellites indicates that the tumor arose between 200 and 2500 years ago.” ⁶ Amazingly enough,  it seemed that the tumor had been passed between dogs perhaps for a couple of thousand years.

But it turns out that it’s even more amazing than that: The 2500 year estimate turns out to be an underestimate. “The estimated time of origin of CTVT is 6500–65,000 years ago. … The cycle of infection currently takes about six months; if this has been true for, say, 15,000 years, then there have been 30,000 transmission events.”⁵ That spans the age where dogs were domesticated and diverged from wolves , about 10,000-15,000 years ago; and in fact, based on genome analysis of the tumors, the tumors did arise in wolves, not dogs. ⁷

This raises a fascinating scenario (which I didn’t see spelled out in Reddeck et al, though I think they were hinting at it). It’s still mysterious how CTVT spreads between hosts. As I said, tumors should act like tissue grafts, which are very rapidly rejected if they’re not MHC matched (as is the case with the CTVT and modern dogs). But tumors could, theoretically, spread within a population that has minimal MHC diversity — that is, an inbred population, that has recently undergone a genetic bottleneck. Such a bottleneck very likely happened during the domestication of dogs, as they evolved from wolves. Was the original CTVT a tumor of one of the very first domesticated proto-dogs — an inbred proto-dog,⁸ a member of a small and inbred population — a spontaneous, fairly ordinary tumor that then spread to the first host’s MHC-matched neighbours in the small and inbred population? That would not be so mysterious; we know tumors can be transplanted between genetically identical hosts, that’s done with lab mice all the time. Then, as the domestic dog population expanded and spread over the world, the tumor would expand and spread along with it, and would have the opportunity to undergo natural selection, adapting to the newly-diverse MHC and to the immune responses that arose after the tumor did. We don’t know, in molecular terms, how the tumor has adapted — we don’t know why it’s able to spread in grow in spite of host immune responses. But at least we can now imagine how the tumor could have reached this point. Thirty thousand transmission events — a couple of million cell divisions — is a long time for a cancer to adapt.

1. Even most viral cancers are probably dead ends, because many viral cancers represent abortive viral replication.[↩]
2. DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history.
   Yuhki N, O’Brien SJ
   Proc Natl Acad Sci USA (1990) 87:836–840.[↩]
3. Molecular Genetic Insights on Cheetah (Acinonyx jubatus) Ecology and Conservation in Namibia
   Laurie L. Marker, Alison J. Pearks Wilkerson, Ronald J. Sarno, Janice Martenson, Christian Breitenmoser-Würsten, Stephen J. O’Brien, and Warren E. Johnson
   Journal of Heredity 2008 99(1):2-13; doi:10.1093/jhered/esm081[↩]
4. Genetic basis for species vulnerability in the cheetah.
   O’Brien SJ, Roelke ME, Marker L, Newman A, Winkler CA, Meltzer D, Colly L, Evermann JF, Bush M, Wildt DE.
   Science. 1985 Mar 22;227(4693):1428-34

   It was suggested at the time, and is widely believed, that cheetahs are particularly susceptible to infectious diseases for this reason, but I think the evidence for this is very weak. Cheetahs have high mortality when infected with feline infectious peritonitis virus, but it’s not unusual for viruses infecting new species to have a high mortality — think SARS and avian influenza in hunans, canine distemper in seals, and feline (or something) parvovirus in dogs, all of which had high mortality in their new, MHC-diverse, host[↩]

5. Rebbeck, C., Thomas, R., Breen, M., Leroi, A., & Burt, A. (2009). ORIGINS AND EVOLUTION OF A TRANSMISSIBLE CANCER Evolution DOI: 10.1111/j.1558-5646.2009.00724.x[↩][↩]
6. Murgia, C., Pritchard, J. K., Kim, S. Y., Fassati, A., and Weiss, R. A. (2006). Clonal origin and evolution of a transmissible cancer. Cell 126, 477-487 .[↩][↩]
7. To be fair to Murgia, they pointed out that their 250-2500 year estimate was for the divergence of the tumor, not its origin, and they specifically raised the possibility that the tumor originated earlier than dogs.[↩]
8. Reddeck et al. point out that the tumor did, in fact, arise from an inbred animal[↩]

You can go to the most prestigious medical center in the world and ask “How is my immune system?” and, after a short period of eye rolling and looks of amused incomprehension, you might (if they don’t just throw you out) be offered a white blood cell count (which you should probably decline). … How did we arrive at this state of affairs? A good case can be made that the mouse has been so successful at uncovering basic immunologic mechanisms that now many immunologists rely on it to answer every question. … Well, except that mice are lousy models for clinical studies. This is readily apparent in autoimmunity (von Herrath and Nepom, 2005) and in cancer immunotherapy (Ostrand-Rosenberg, 2004), where of dozens (if not hundreds) of protocols that work well in mice, very few have been successful in humans. ¹

Cytotoxic T lymphocyte attacking a tumor cell

The above (emphasis added) is from a recent manifesto from immunology giant Mark Davis.¹ (He isn’t the first to make this point, of course, but when Mark Davis speaks, immunologists listen.)  Davis’s suggested solution was, among other things, to start using humans as their own models, taking advantage of the large numbers of humans who are routinely screened and overcoming the lack of experimental control by taking a “systems” approach, including large-scale data collection from healthy and ill people and large-scale informatics as part of the analysis.² (He also comments on the “humanized” mouse approach that I mentioned briefly the other day.)

Here’s an example of the power of human models, though it’s not exactly what Davis is describing.³  I’ve mentioned before the evidence that cancers in mice are controlled by the immune system (for example, here and links therein).  In those experiments, mutant mice, lacking one or more components of the immune response, were shown to be predisposed to cancer.  There are also a couple of human studies that indicate the same thing; people on long-term immunosuppression (as in transplant recipients) are somewhat more likely to get certain kinds of cancer, for example.

CTL attacking a tumor cell

One advantage of using humans as models of their own diseases is that there are an amazing number of well-documented mutations and disease-associated genes in humans.  Human disease is taken very seriously, it’s well funded (at least in comparison to, say, dog and cat disease); even diseases that are very rare can be identified in humans and the gene variant identified.  One such disease is Type II familial hemophagocytic lymphohistiocytosis (FHL), a rare, rapidly fatal disease caused by mutations in the perforin gene. ⁴  Perforin is important in cytotoxic T lymphocyte function, and it’s one of the immune genes that has been shown to be important in preventing cancers in mice.⁵

Are patients with Type II FHL at risk of developing cancer, like mice with targeted perforin mutations?  It’s not an easy question to ask, because most patients die relatively young.  But because these are humans, this very rare disease is nevertheless well documented, and the authors were able to search the literature for a suitable subset of patients:

… we identified a subgroup of individuals from nonconsanguineous families who possessed 2 mutated PRF1 alleles but whose onset of FHL was markedly delayed (the age at onset of 10 years or older) or even abolished. A total of only 23 such cases could be identified in the entire literature …  Ten of the individuals (Patients 14–23 inTable 1) developed manifestations of FHL without any other significant infectious or neoplastic sequelae reported. … Remarkably, in 11 of these 13 individuals (or 48% of the entire cohort of 23), the primary clinical presentation was with either B or T cell lymphoma or acute or chronic leukemia of lymphoid origin. … The very high frequency of hematological cancers in this 23-patient cohort …  is vastly in excess of that in the general population.³

There’s a lot of other interesting stuff in the paper, but this is enough to make the point: Just as in mice, perforin in humans (and therefore, the immune system) is important in preventing cancer.

I’ll leave with this now-familiar observation from the paper:

It is clearly problematic to extrapolate experimental data from inbred mouse strains to an outbred human setting where such evidence is far more difficult to gather.³

1. DAVIS, M. (2008). A Prescription for Human Immunology Immunity, 29 (6), 835-838 DOI: 10.1016/j.immuni.2008.12.003[↩][↩]
2. Again, of course, Davis isn’t the first to advocate this approach.[↩]
3. Chia, J., Yeo, K., Whisstock, J., Dunstone, M., Trapani, J., & Voskoboinik, I. (2009). Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer Proceedings of the National Academy of Sciences, 106 (24), 9809-9814 DOI: 10.1073/pnas.0903815106[↩][↩][↩]
4. Familial hemophagocytic lymphohistiocytosis. Primary hemophagocytic lymphohistiocytosis.
   Henter JI, Aricò M, Elinder G, Imashuku S, Janka G.
   Hematol Oncol Clin North Am. 1998 Apr;12(2):417-33[↩]
5. Perforin-mediated Cytotoxicity Is Critical for Surveillance of Spontaneous Lymphoma.
   Mark J. Smyth, Kevin Y.T. Thia, Shayna E.A. Street, Duncan MacGregor, Dale I. Godfrey, and Joseph A. Trapani.
   The Journal of Experimental Medicine, Volume 192, Number 5, September 5, 2000 755-760[↩]

Caco2 colon carcinoma cells

One of my long-standing questions now has at least a partial answer, or maybe a pathway toward a partial answer.

My question was, “Why are different tumors the same?” That is, why do tumors of the same type often seem to have similar immunological changes?

Viruses of the same kind (all herpes simplex viruses, say) all avoid immunity in the same way because they all have a common ancestor from which they inherited their immune evasion molecules. But tumors have no common ancestor; each tumor has to grope around and find its own solution to common problems independently. So why should tumors of a particular tissue converge on common solutions? What would make a certain pathway a simple solution for colon carcinomas, but not for bladder tumors? And yet, apparently that’s what happens. Even though many different tumor types become non-immunogenic, tumors of a particular tissue type often reach non-immunogenicity by a similar path. For example:

… distinct molecular events underlie HLA class I loss, depending on the aetiology of the tumours; Lynch syndrome-related cancers presented with mutations in the β₂-m molecule, while sporadic microsatellite-unstable tumours mainly showed alterations in the antigen-processing machinery components ¹

When I posed this question a couple of months ago, I didn’t have an answer. I said

Part of the answer may be that the particular oncogenes associated with different tumor types lead to particular transcriptional hot-spots, and being a transcriptional hot-spot makes the region a mutational hot-spot as well, but at least as I understand it that’s not enough to account for the trends.

EH, in the comments, suggested a couple more possibilities: Perhaps destruction of a particular pathway is “a nice side benefit of destroying some yet undiscovered tumor suppressor important for melanoma or colon cancer? Or maybe the loss of certain repair genes common to a cancer type (like BRCA) leads to selective chromosomal instability in parts of chromosome 6?“

It turns out, unsurprisingly, that I’m not the only one to ask the question. There is no general answer, but apparently a partial answer has been kicking around for a while now, and Hans Morreau’s group is starting to put some of the pieces together.² As their model, Morreau’s group looked at MUTYH-associated polyposis (“MAP”)-associated tumors. (MAP is a heritable defect in DNA repair, so MAP patients have high rates of mutagenesis and usually develop colon carcinomas.) They reasoned that

MAP tumours could be more prone to stimulate a cytotoxic T-cell-mediated immune response, due to their frequent generation of aberrant peptides. Hence, these tumours could also be subjected to a strong selective pressure favouring the outgrowth of cancer cells that acquire an immune evasive phenotype.

DNA Repair

In other words, the argument is that the group of tumors that lose (or reduce) DNA repair are much more likely to throw out mutant proteins. These mutant proteins are targets for the immune response (because they’re no longer “self” antigens), so to survive the immune attack the tumor has a strong selection for loss of immunogenicity (and also has the high mutation rate that allows them to rapidly mutate away from immunogenicity).

Sure enough, the tumors did frequently (72%) have defects in antigen presentation. (In fact, because of the way they measured HLA expression — by immunohistochemistry rather than sequencing — I would bet that the rate of functional defects was actually much higher than that.) They conclude that this “provides additional evidence that tumours carrying defects in DNA base repair mechanisms are more prone to undergo immune escape mechanisms.” ²  Since they don’t formally compare to other tumor types, I don’t think they can really say “more prone” — you’d have to use the same techniques to look at tumors from non-MAP patients to be able to say that. Still, I do think that is a significantly higher rate than has been turned up in previous studies using similar techniques,³ so I’ll tentatively accept that conclusion.

This doesn’t really explain why similar tumors target similar components, but it’s at least a conceptual connection between different tumor types and an underlying pathway. I’d be interested in a more large-scale screen, looking at cancers with stronger and weaker mutator phenotypes to see if common pathways emerge. One may already have popped up, since the authors note here that expression of β₂-m was frequently lost⁴ in several tumors with DNA repair defects:

Although speculative, it is interesting to underline that carcinomas derived from both MAP and Lynch syndromes preferentially lose β₂-m expression coupled to HLA class I deficiencies. A functional explanation for these observations remains elusive, but perhaps distinct reactions (both qualitative and quantitative) by the immune system, depending on the age of onset of the tumours, could condition the type of mechanisms that lead to HLA class I expression deficiencies. ²

Again, we would need to compare to other tumor types to see if this really is more frequent, but overall it feels as if there’s a hint of a pathway here. At least there are some specific questions that can be asked.

1. de Miranda, N., Nielsen, M., Pereira, D., van Puijenbroek, M., Vasen, H., Hes, F., van Wezel, T., & Morreau, H. (2009). MUTYH-associated polyposis carcinomas frequently lose HLA class I expression-a common event amongst DNA-repair-deficient colorectal cancers The Journal of Pathology DOI: 10.1002/path.2569
   Referencing
   Dierssen JWF, de Miranda NFCC, Ferrone S, van Puijenbroek M, Cornelisse CJ, Fleuren GJ, et al. HNPCC versus sporadic microsatellite-unstable colon cancers follow different routes toward loss of HLA class I expression. BMC Cancer 2007; 7: 33[↩]
2. de Miranda, N., Nielsen, M., Pereira, D., van Puijenbroek, M., Vasen, H., Hes, F., van Wezel, T., & Morreau, H. (2009). MUTYH-associated polyposis carcinomas frequently lose HLA class I expression-a common event amongst DNA-repair-deficient colorectal cancers The Journal of Pathology DOI: 10.1002/path.2569[↩][↩][↩]
3. Other studies, especially those of Soldano Ferrone, have turned up much higher rates of functional HLA class I defects, but they’ve used more focused, and more difficult and expensive, techniques to reach that conclusion.[↩]
4. β₂-m is a physical component of the MHC class I complex[↩]

We know that the immune system can destroy tumors. We also know, unfortunately, that by the time we see a tumor, immunity probably won’t destroy the tumor. There are lots of reasons for that. One is that tumors are essentially part of the normal body, so it’s normal for the immune system to ignore them. It looks as if you need to have immunity that’s just right to get rid of a tumor.

Tumors arise from normal self cells,¹ that the immune response has been programmed to ignore. Now, the process of becoming a tumor is not normal, and so tumors are not entirely normal self any more — meaning that there are likely to be some targets in most if not all tumors. But in all but the most reckless tumors the differences between abnormal and normal are relatively small, compared to, say, a virus-infected cell that contains many potential targets.

There’s actually a long list of known tumor antigens; the T-cell tumor peptide database lists many hundreds of them. But most are not truly specific for the tumor.  The’re actually normal self antigens; they’re derived from proteins that are overexpressed in tumors, or that are differentiation antigens or “cancer-germline” antigens that are normally also found in self tissues. What’s more, these normal self antigens are the most interesting tumor antigens, as far as clinical utility is concerned. Mutations can make brand-new, non-self targets for the immune system, but they’re going to be sporadic targets, often unique to individual tumors — not something you can prepare for. The normal antigens, though, are likely to be predictable, common targets; it’s conceivable that tumor vaccines can be prepared in advance.

Human melanoma cell

If these antigens were common (which they are, in some tumor types — like melanoma), and they were good targets for the immune system, then we wouldn’t see much cancer. We do see melanomas quite often, and part of the reason may be that the immune system generally responds quite weakly to these antigens.  Why is that? And, more to the point, how can we make the immune system respond more strongly? A recent paper in the Journal of Experimental Medicine² offers answers for both of these questions.

From work in the past couple of years, we now have decent estimates of how many T cells there are that can react with any particular target. (See here and here for my discussion of the earlier papers.) A reasonably strong immune response to a non-self epitope might originate from maybe 100 or so precursor T cells. There’s a rather wide range of frequency for these precursor cells, say from 20 to 1000; and to some extent, the fewer T cells there are the weaker (the less immunodominant) the immune response.

We expect T cells against normal self targets to be less common, because they should be eliminated as they mature in the thymus. Some may survive, though, and we would count on these survivors to attack the normal (albeit overexpressed, or abnormally present) target in the cancer cells. But just how rare are they?

Rizzuto et al say they’re really rare (this was in mice, by the way); at least ten times less abundant than T cells against non-self antigens.  If you look at the range I gave for “normal” precursors, that could mean there are fewer than 5 or 10 precursors.  If the average is “fewer than five”, then quite possibly some mice have only two, or one, or no precursors.  You can’t have much of a response with no precursors.

So there’s a weak anti-tumor response because there aren’t many T cells in the body that can respond to the normal self targets in the tumor. That’s not really a surprise, but it does raise the question, What if there were more of the T cells? To ask that question, Rizzuto et al. tried transferring more of these precursor T cells into tumor-bearing mice — starting at around the normal level for a precursor to non-self antigen, and going up from there — and then vaccinating with the appropriate target.

The effects were pretty dramatic. With no supplemental T cells (that is, with the natural, very low, level of T cell precursors) the mice all died of the tumor quickly. At the middle of the range, almost all of the mice rejected the tumor. And at the highest levels of transfers? The mice all died again. Having enough T cells to respond was protective, but putting in too many made them useless.

These results identify vaccine-specific CD8+ precursor frequency as a remarkably significant predictor of treatment and side-effect outcome. Paradoxically, above a certain threshold there is an inverse relationship between pmel-1 clonal frequency and vaccine-induced tumor rejection.²

Mouse melanoma cell

(My emphasis) This paradoxical effect is probably because the numerous T cells started to compete with each other so that none of them were properly activated; they only saw effective-looking polyfunctional T cells at the lower transfer levels.

In other words, if you’re going to transfer T cells to try to eliminate a tumor, more is not necessarily better. Quality and quantity are both important factors, and quantity helps determine quality.

One question I have is how this relates to tumor immune evasion. Many tumor types  acquire mutations, as they develop, that block presentation of antigen to T cells. Are these mutations perhaps only partially effective — giving the tumors sufficient protection against the tiny handful of natural precursors they “expect” to deal with, but not against a larger attack after, say, vaccination — or are they more complete, and protective even if the optimal number of T cells are transfered? I’d guess that it would depend on the tumor, but it looks as if it might be a relevant question and it would be nice to have more than a guess.

Our results show that combining lymphodepletion with physiologically relevant numbers of naive tumor-specific CD8+ cells and in vivo administration of an effective vaccine generates a high-quality, antitumor response in mice. This approach requires strikingly low numbers of naive tumor-specific cells, making it a new and truly potent treatment strategy.   ²

1. I’m ignoring here crazy things like the contagious tumors of Tasmanian Devils and dogs[↩]
2. Rizzuto, G., Merghoub, T., Hirschhorn-Cymerman, D., Liu, C., Lesokhin, A., Sahawneh, D., Zhong, H., Panageas, K., Perales, M., Altan-Bonnet, G., Wolchok, J., & Houghton, A. (2009). Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response Journal of Experimental Medicine, 206 (4), 849-866 DOI: 10.1084/jem.20081382[↩][↩][↩]

Hierarchical clustering of breast carcinomas1

Something that’s puzzled me for years is why the same kinds of tumors tend to have the same kinds of immune evasion mechanisms. And I’m not going to give an answer, just trying to share the confusion a little.

What I mean is this:

It has been demonstrated that human tumors of distinct histology express low or downregulated MHC class I surface antigens … The distinct frequency of MHC class I abnormalities is caused by total HLA class I antigen loss, HLA class I down-regulation as well as loss or down-regulation of HLA class I allo-specificities. However, the frequency and mode of these defects significantly varied between the types of tumors analysed and could be associated in some cases with microsatellite instability. ²

(My emphasis) As I’ve noted here several times (most specifically here) tumors very often evade the immune system as they mature. Cytotoxic T lymphocytes (CTL) can control tumors in the tumors’ eary stages, but by the time we detect a tumor clinically the tumor is almost always resistant to the immune system. They do this in various ways, including inducing regulatory T cells, but also by mutating themselves to make themselves invisible to CTL (and other components of the immune system, but let’s keep it simpler for the moment).

There are a myriad ways for a tumor to become invisible, at the molecular level.  The MHC class I antigen presentation pathway is long and complex, and for any partiuclar tumor there are likely to be many different bottlenecks, points of attack.  Since tumors are all independent events³, so at first, and even second, glance, there’s no obvious reason why tumors of the same type should find a similar approach.  That is, just because two colon carcinomas look the same histologically in two different individuals, there’s no link between them.  ⁴ Why should colon carcinomas avoid CTL using one set of mutations, while, say, breast cancers use a different set of mutations? Yet apparently, that’s what tends to happen; for example:

Mutations or deletions in β2-m were detected in colon carcinoma (21%), melanoma (15%) and other tumors (<5%). So far, no mutations in β2-m have been found in RCC lesions, bladder and laryngeal tumors despite MHC class I loss or downregulation. … haplotype loss was found in head and neck squamous cell carcinoma (HNSCC) with a frequency of 36%, whereas in renal cell carcinoma (RCC) LOH only occurs in approximately 12% of tumor lesions analyzed. ²

If we saw these patterns only with virus-associated cancers, such as cervical carcinomas and even hepatic carcinomas, there would at least be a common link, but these tumors are not (as far as we know) caused by viruses in humans.

Part of the answer may be that the particular oncogenes associated with different tumor types lead to particular transcriptional hot-spots, and being a transcriptional hot-spot makes the region a mutational hot-spot as well, but at least as I understand it that’s not enough to account for the trends.

So why are particular MHC abnormalities linked to tumor type?  Anyone?

1. Turashvili et al. BMC Cancer 2007 7:55   doi:10.1186/1471-2407-7-55[↩]
2. Seliger, B. (2008). Molecular mechanisms of MHC class I abnormalities and APM components in human tumors Cancer Immunology, Immunotherapy, 57 (11), 1719-1726 DOI: 10.1007/s00262-008-0515-4[↩][↩]
3. barring such weird things as canine transmissible venereal tumor and Tasmanian Devil facial tumors; see here for more on those[↩]
4. The comparison is, of course, viruses.  A herpesvirus of chickens, and one of humans, may both use immune evasion mechanisms, but they have a common ancestor even if it’s a couple of hundred million years ago.[↩]

Metchnikov: “Lecons sur la pathologie” (1892)

There are times when you just feel like the universe is out to get you. For example, we know that inflammation can drive tumor formation; but a paper just came out that suggests reducing inflammation can also drive tumor formation. ¹ It doesn’t seem fair.

I’ve previously mentioned the link between inflammation and tumorigenesis, which is probably at least partly because the inflammation produces reactive oxygen and nitrogen species (RONS ) that are tumorigenic.

I’ve also talked about the link between reduced inflammation and ongoing tumors (for example, here, here, and here). What seems to be going on here is that regulatory T cells (TRegs) are induced by tumors, and these TRegs shut down anti-tumor immunity.

So far, these findings aren’t really contradictory. Increasing inflammation before a tumor is present makes tumors more likely to form. After the tumor has formed, reducing inflammation makes the tumor more likely to persist. The universe-is-against-us part comes from the suggestion that reducing inflammation (via TRegs) before tumor formation, also makes the tumors more likely to form.

This may be a special case. The paper from Philip Dennis’s group ¹ looked at a specific set of cancers, those associated with K-Ras mutations (linked to smoking-induced lung cancer). K-Ras activation itself triggers inflammation (for reasons I, at any rate, don’t understand). When K-Ras is activated, as well as inflammation, TRegs move into the area, and presumably reduce the inflammation. Depleting the TRegs (and therefore increasing the inflammation) decreased the number of tumors by 75% — the opposite of what you’d expect if inflammatory RONS were driving tumorigenesis.

A common feature linking smoking induced K-Ras mutations in human lung cancer and preclinical models driven by tobacco carcinogens that cause K-Ras mutations is inflammation. In both cases, the presence of Foxp3+ cells is likely important for limiting the extent of inflammation and tissue damage, albeit at a potential cost of promoting tumorigenesis. ¹

In later-stage tumors the situation became more consistent with other work — getting rid of TRegs reduced the tumors, suggesting that these tumors were depending on TRegs to prevent immune clearance:

Aggressive and invasive K-Ras-induced adenocarcinomas (IO33 and K-RasLA2) remained sensitive to more direct targeting of Foxp3+ cells through a neutralizing anti-CD25 antibody or genetic deletion. This indicates that direct Treg cell depletion strategies that are being evaluated clinically could have therapeutic value in more advanced stages of K-Ras driven lung cancer. ¹

My question here is whether the early inflammation is kind of a red herring. Could the TReg depletion in the early stages be reducing the anti-tumor immune response in a specific way, just as in the later stages of tumor formation? That is, could the TReg depletion lead to a tumor-specific immune response, which prevents tumors from forming? In this case the inflammation could still be driving the tumor formation, but the increase in tumor formation would be outweighed by the simultaneous increase in anti-tumor immunity. I don’t know quite how to test this, but perhaps doing the same experiment in mice lacking, say, CD8 T cells might be interesting. (Such mice should still have the early inflammation and the TRegs, but may have a less effective immune response. It’s not a perfect experiment, though, for reasons that are probably too complex to go into here.)

1. Granville, C., Memmott, R., Balogh, A., Mariotti, J., Kawabata, S., Han, W., LoPiccolo, J., Foley, J., Liewehr, D., Steinberg, S., Fowler, D., Hollander, M., & Dennis, P. (2009). A Central Role for Foxp3+ Regulatory T Cells in K-Ras-Driven Lung Tumorigenesis PLoS ONE, 4 (3) DOI: 10.1371/journal.pone.0005061[↩][↩][↩][↩]