Sticky Cancer Genes

 

In the previous blog I talked about Breath Biopsy — a new method that aims to detect cancers from breath samples. I noted that it could end up complementing liquid biopsies — samples of tumour cell DNA pulled out of a teaspoon of blood — both being, as near as makes no difference, non-invasive tests. Just to show that there’s no limit to the ingenuity of scientists, yet another approach to the detection problem has just been published — this from Matt Trau and his wonderful team at The University of Queensland.

This new method, like the liquid biopsy, detects DNA but, rather than the sequence of bases, it identifies an epigenetic profile — that is, the pattern of chemical tags (methyl groups) attached to bases. As we noted in Cancer GPS? cancer cells often have abnormal DNA methylation patterns — excess methylation (hypermethylation) in some regions, reduced methylation in others. Methylation acts as a kind of ‘fine tuner’, regulating whether genes are switched on or off. In the methylation landscape of cancer cells there is an overall loss of methylation but there’s an increase in regions that regulate the expression of critical genes. This shows up as clusters of methylated cytosine bases.

Rather helpfully, a little while ago in Desperately SEEKing … we talked about epigenetics and included a scheme showing how differences in methylation clusters can identify normal cells and a variety of cancers and how these were analysed in the computer program CancerLocator.

The Trau paper has an even better scheme showing how the patterns of DNA decoration differ between normal and cancer cells and how this ‘methylscape’ (methylation landscape) affects the physical behaviour of DNA.

How epigenetic changes affect DNA. Scheme shows methylation (left: addition of a methyl group to a cytosine base in DNA) in the process of epigenetic reprogramming in cancer cells. This change in the methylation landscape affects the solubility of DNA and its adsorption by gold (from Sina et al. 2018).

Critically, normal and cancer epigenomes differ in stickiness — affinity — for metal surfaces, in particular for gold. In a clever ploy this work incorporated a colour change as indicator. We don’t need to bother with the details — and the result is easy to describe. DNA, extracted from a small blood sample, is added to water containing tiny gold nanoparticles. The colour indicator makes the water pink. If the DNA is from cancer cells the water retains its original colour. If it’s normal DNA from healthy cells the different binding properties turns the water blue.

By this test the Brisbane group have been able to identify DNA from breast, prostate and colorectal cancers as well as from lymphomas.

So effective is this method that it can detect circulating free DNA from tumour cells within 10 minutes of taking a blood sample.

The aim of the game — and the reason why so much effort is being expended — is to detect cancers much earlier than current methods (mammography, etc.) can manage. The idea is that if we can do this not weeks or months but perhaps years earlier, at that stage cancers may be much more susceptible to drug treatments. Trau’s paper notes that their test is sensitive enough to detect very low levels of cancer DNA — not the same thing as early detection but suggestive none the less.

So there are now at least three non-invasive tests for cancer: liquid biopsy, Breath Biopsy and the Queensland group’s Methylscape, and in the context of epigenetics we should also bear in mind the CancerLocator analysis programme.

Matt Trau acknowledges, speaking of Methylscape, that “We certainly don’t know yet whether it’s the holy grail for all cancer diagnostics, but it looks really interesting as an incredibly simple universal marker for cancer …” We know already that liquid biopsies can give useful information about patient response to treatment but it will be a while before we can determine how far back any of these methods can push the detection frontier. In the meantime it would be surprising if these tests were not being applied to age-grouped sets of normal individuals — because one would expect the frequency of cancer indication to be lower in younger people.

From a scientific point of view it would be exciting if a significant proportion of ‘positives’ was detected in, say, 20 to 30 year olds. Such a result would, however, raise some very tricky questions in terms of what, at the moment, should be done with those findings.

Reference

Abu Ali Ibn Sina, Laura G. Carrascosa, Ziyu Liang, Yadveer S. Grewal, Andri Wardiana, Muhammad J. A. Shiddiky, Robert A. Gardiner, Hemamali Samaratunga, Maher K. Gandhi, Rodney J. Scott, Darren Korbie & Matt Trau (2018). Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker. Nature Communications 9, Article number: 4915.

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Keeping Up With Cancer

 

Cancer enthusiasts will know that there are zillions of web sites giving info on cancer stats — incidence, mortality, etc. — around the world. Notable is the World Health Organization’s Globocan, an amazing compilation of data on all cancers from every country. The Global Burden of Disease Cancer Collaboration audits diagnosis rates and deaths for 29 types of cancer around the world each year. Needless to say, this too is a vast undertaking involving hundreds of scientists around the world. The organizing genius is Dr. Christina Fitzmaurice of the Institute for Health Metrics and Evaluation in the University of Washington, Seattle and, under her guidance, their update for 2016 has just come out.

What’s new?

In 2016 there were 17.2 million people diagnosed with cancer. 8.9 million died from cancers. By 2030 the number of new cancer cases per year is expected to reach 24 million. Well, you knew the numbers were going to be big — almost incomprehensibly so. But here’s the real shaker: the 17.2 M is 28% up on the 2006 figure — yes, that’s a rise of more than one quarter.

Yearly global cancer deaths from 1990 to 2016.

The green line is total deaths per 100,000 people.

Red line: Cancer death rates taking account of the increase in world population.

Blue line: Age-standardized death rates: these are corrected for population size and age structure. Age-standardization therefore gives a better indication of the prevalence and incidence of underlying cancer risk factors between countries and with time without the influence of demographic and population structure changes.

The numbers on the vertical axis are deaths per 100,000 people. From Our World in Data.

Any real surprises?

No. An increase of more than one quarter in cancer cases does indeed make you think but the grim numbers are only what you would predict from looking at the trends over the last 40 years. The graph shows death rates that, of course, reflect incidence. The total figure (top) shows starkly how the rise in the population of the world and our increasing life-span is steadily pushing up the overall cancer burden.

So it’s a mega-problem but the trends are smooth and gradual. There’s been no drastic upheaval.

Global trends

Yearly global cancer deaths from 1990 to 2016 for the four major cancer types.

The numbers on the vertical axis are age-standardized death rates per 100,000 people.

Because age-standardization assumes a constant population age & structure it permits comparisons between countries and over time without the effects of a changing age distribution within a population. From Our World in Data.

The global trends in deaths from the four major cancers look mildly encouraging (above). However, these should not cheer us up too much. In the developed world there are some positives. In the USA, for example, over the last 17 years deaths from prostate are down from 31.6 to 18.9, for breast from 26.6 to 20.3 and for lung from 55.4 to 40.6 per 100,000 people. For bowel cancer there’s been a slight increase (4.1 to 4.8).

In the wider world, however, the really dispiriting thing shown by the latest figures is that the increases in incidence and deaths are greatest in low- and middle-income countries.

What can we do?

Lung cancer (includes cancers of the trachea and bronchi) remains the world’s biggest cancer killer, accounting for 20% of all deaths in 2016. Over 90% of these were caused by tobacco. In the UK and the USA lung cancer deaths in men have markedly declined as a result of widespread smoking bans, as the graph below shows, and the female figures have started to show a sight decline.

Lung cancer deaths per 100,000 by sex from 1950 to 2002 for the UK and the USA. From Our World in Data.

Asia contributes over half the global burden of cancer but the incidence in Asia is about half that in North America. However, the ratio of cancer deaths to the number of new cancer cases in Asia is double that in North America. Although the leading cause of death world-wide is heart disease, in China it is cancer. Every year more than four million Chinese are diagnosed with the disease and nearly three million die from it. Overall, tobacco smoking is responsible for about one-quarter of all cancer deaths in China. Nevertheless, Chinese smoking rates continue to rise and air pollution in the major cities is fuelling the problem.

The under-developed world, however, continues to be targeted by the tobacco industry and the successful promotion of their products means that there is no end in sight to one of mankind’s more bizarre and revolting forms of self-destruction.

There are, of course, other things within our control that contribute significantly to the global cancer burden. If only we could give everyone clean water to drink, restrict our red meat and processed food consumption and control our exposure to uv in sunlight we would cut cancer by at least one half.

If only …

Reference

The Global Burden of Disease Cancer Collaboration (2018). Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-Years for 29 Cancer Groups, 1990 to 2016: A Systematic Analysis for the Global Burden of Disease Study. JAMA Oncol. Published online June 2, 2018. doi:10.1001/jamaoncol.2018.2706.

No It Isn’t!

 

It’s great that newspapers carry the number of science items they do but, as regular readers will know, there’s nothing like the typical cancer headline to get me squawking ‘No it isn’t!” Step forward The Independent with the latest: “Major breakthrough in cancer care … groundbreaking international collaboration …”

Let’s be clear: the subject usually is interesting. In this case it certainly is and it deserves better headlines.

So what has happened?

A big flurry of research papers has just emerged from a joint project of the National Cancer Institute and the National Human Genome Research Institute to make something called The Cancer Genome Atlas (TCGA). This massive initiative is, of course, an offspring of the Human Genome Project, the first full sequencing of the 3,000 million base-pairs of human DNA, completed in 2003. The intervening 15 years have seen a technical revolution, perhaps unparalled in the history of science, such that now genomes can be sequenced in an hour or two for a few hundred dollars. TCGA began in 2006 with the aim of providing a genetic data-base for three cancer types: lung, ovarian, and glioblastoma. Such was its success that it soon expanded to a vast, comprehensive dataset of more than 11,000 cases across 33 tumor types, describing the variety of molecular changes that drive the cancers. The upshot is now being called the Pan-Cancer Atlas — PanCan Atlas, for short.

What do we need to know?

Fortunately not much of the humungous amounts of detail but the scheme below gives an inkling of the scale of this wonderful endeavour — it’s from a short, very readable summary by Carolyn Hutter and Jean Claude Zenklusen.

TCGA by numbers. The scale of the effort and output from The Cancer Genome Atlas. From Hutter and Zenklusen, 2018.

The first point is obvious: sequencing 11,000 paired tumour and normal tissue samples produced mind-boggling masses of data. 2.5 petabytes, in fact. If you have to think twice about your gigas and teras, 1 PB = 1,000,000,000,000,000 B, i.e. 1015 B or 1000 terabytes. A PB is sometimes called, apparently, a quadrillion — and, as the scheme helpfully notes, you’d need over 200,000 DVDs to store it.

The 33 different tumour types included all the common cancers (breast, bowel, lung, prostate, etc.) and 10 rare types.

The figure of seven data types refers to the variety of information accumulated in these studies (e.g., mutations that affect genes, epigenetic changes (DNA methylation), RNA and protein expression, duplication or deletion of stretches of DNA (copy number variation), etc.

After which it’s worth pausing for a moment to contemplate the effort and organization involved in collecting 11,000 paired samples, sequencing them and analyzing the output. It’s true that sequencing itself is now fairly routine, but that’s still an awful lot of experiments. But think for even longer about what’s gone into making some kind of sense of the monstrous amount of data generated.

And it’s important because?

The findings confirm a trend that has begun to emerge over the last few years, namely that the classification of cancers is being redefined. Traditionally they have been grouped on the basis of the tissue of origin (breast, bowel, etc.) but this will gradually be replaced by genetic grouping, reflecting the fact that seemingly unrelated cancers can be driven by common pathways.

The most encouraging thing to come out of the genetic changes driving these tumours is that for about half of them potential treatments are already available. That’s quite a surprise but it doesn’t mean that hitting those targets will actually work as anti-cancer strategies. Nevertheless, it’s a cheering point that the output of this phenomenal project may, as one of the papers noted, serve as a launching pad for real benefit in the not too distant future.

What should science journalists do to stop upsetting me?

Read the papers they comment on rather than simply relying on press releases, never use the words ‘breakthrough’ or ‘groundbreaking’ and grasp the point that science proceeds in very small steps, not always forward, governed by available methods. This work is quite staggering for it is on a scale that is close to unimaginable and, in the end, it will lead to treatments that will affect the lives of almost everyone — but it is just another example of science doing what science does.

References

Hutter, C. and Zenklusen, J.C. (2018). The Cancer Genome Atlas: Creating Lasting Value beyond Its Data. Cell 173, 283–285.

Hoadley, K.A. et al. (2018). Cell-of-Origin Patterns Dominate the Molecular Classification of 10,000 Tumors from 33 Types of Cancer. Cell 173, 291–304.

Hoadley, K.A. et al. (2014). Multiplatform Analysis of 12 Cancer Types Reveals Molecular Classification within and across Tissues of Origin. Cell 158, 929–944.

Please … Not Another Helping

 

You may have seen the headlines of the: “Processed food, sugary cereals and sliced bread may contribute to cancer risk” ilk, as this recently published study (February 2018) was extensively covered in the media — the Times of London had a front page spread no less.

So I feel obliged to follow suit — albeit with a heavy heart: it’s one of those depressing exercises in which you’re sure you know the answer before you start.

Who dunnit?

It’s a mainly French study (well, it is about food) led by Thibault Fiolet, Mathilde Touvier and colleagues from the Sorbonne in Paris. It’s what’s called a prospective cohort study, meaning that a group of individuals, who in this case differed in what they ate, were followed over time to see if diet affected their risk of getting cancers and in particular whether it had any impact on breast, prostate or colorectal cancer. They started acquiring participants about 20 years ago and their report in the British Medical Journal summarized how nearly 105 thousand French adults got on consuming 3,300 (!) different food items between them, based on each person keeping 24 hour dietary records designed to record their usual consumption.

Foods were grouped according to degree of processing. The stuff under the spotlight is ‘ultra-processed’ — meaning that it has been chemically tinkered with to get rid of bugs, give it a long shelf-life, make it convenient to use, look good and taste palatable.

What makes a food ‘ultra-processed’ is worked out by something called the NOVA classification. I’ve included their categories at the end.

Relative contribution of each food group to ultra-processed food consumption in diet (from Fiolet et al. 2018).

And the result?

The first thing to be said is that this study is a massive labour of love. You need the huge number of over 100,000 cases even to begin to squeeze out statistically significant effects — so the team has put in a terrific amount of work.

After all the squeezing there emerged a marginal increase in risk of getting cancer in the ultra-processed food eaters and a similar slight increase specifically for breast cancer (the hazard ratios were 1.12 and 1.11 respectively). There was no significant link to prostate and colorectal cancers.

Which may mean something. But it’s hard to get excited, not merely because the effects described are small but more so because such studies are desperately fraught and the upshot familiar.

One problem is that they rely on individuals keeping accurate records. Another problem here is that the classification of ‘ultra-processed’ is somewhat arbitrary — and it’s also very broad — leaving one asking what the underlying cause might be: ‘is it sugar, fat or what?’ Furthermore, although the authors tried manfully to allow for factors like smoking and obesity, it’s impossible to do this with complete certainty. The authors themselves noted that, for example, they couldn’t allow for the effects of oral contraception.

The authors are quite right to point out that it is important to disentangle the facets of food processing that bear on our long-term health and that further studies are needed.

I would only add ‘rather you than me.’

Perforce in these pages we have gone on about diets good and bad so there is no need to regurgitate. Suffice to say that my advice on what to eat is the same as that of any other sane person and summarized in Dennis’s Pet Menace — and it’s not been remotely affected by this new research which, in effect, says ‘junk food is probably bad for you in the long run.’ But let’s leave the last word to Tom Sanders of King’s College London: “What people eat is an expression of their life-style in general, and may not be causatively linked to the risk of cancer.” 

Reference

Fiolet, T. et al. (2018). Consumption of ultra-processed foods and cancer risk: results from NutriNet-Santé prospective cohort. BMJ 2018;360:k322 http://dx.doi.org/10.1136/bmj.k322

NOVA classification:

The ultra-processed food group is defined by opposition to the other NOVA groups: “unprocessed or minimally processed foods” (fresh, dried, ground, chilled, frozen, pasteurised, or fermented staple foods such as fruits, vegetables, pulses, rice, pasta, eggs, meat, fish, or milk), “processed culinary ingredients” (salt, vegetable oils, butter, sugar, and other substances extracted from foods and used in kitchens to transform unprocessed or minimally processed foods into culinary preparations), and “processed foods” (canned vegetables with added salt, sugar coated dried fruits, meat products preserved only by salting, cheeses, freshly made unpackaged breads, and other products manufactured with the addition of salt, sugar, or other substances of the “processed culinary ingredients” group).

Much Ado About … Some Things

Given that the ‘festive season’ is approaching, maybe we should try to find something joyous to say about cancer. It’s not difficult. Over the last 60 years (1950-2013) the 5-year Relative Survival Rates for white Americans for breast and prostate cancers have gone from about 50% to over 90% (99.6% in fact for prostate). A number of other types (e.g., testicular cancer) are now largely curable, if treated early enough. Similar trends have occurred in most developed countries – all this through advances in surgery and radiotherapy but, most of all, because of new drugs.

Big Pharma

It’s big business. According to the Financial Times, annual spending on cancer drugs hit $100 billion worldwide in 2014 and is projected to exceed $150 billion by 2020. As you would hope, this expenditure on drug development and production has resulted in a gradual rise in available cancer drugs, represented below by the number of new cancer drugs approved each year by the American Food and Drug Administration (FDA).

Number of new cancer drugs approved each year by the American Food and Drug Administration from 1949 to 2016 (from Hope Cristol, The American Cancer Society, 2016).

Data compiled from drugs@fda.gov, National Cancer Institute, FDA Orange Book, FDA.gov, and centerwatch.com. Reporting and analysis by Sabrina Singleton, ACS research historian.

We should note that the FDA equivalent on this side of the Atlantic is the European Medicines Agency (EMA) and they tend to follow similar licensing patterns. Thus in 2016 a total of 74 new drug approvals were granted by the FDA and the EMA — 19 by the EMA only, 19 by only the FDA, with 36 approved by both. Of the drugs approved by the EMA in 2016, 17 had received prior FDA approval (i.e. in 2015 or earlier). However, only six drugs registered in the US in 2016 had prior EMA approval, indicating that drug companies tend to apply for approval in the US first before registering their products in the EU.

So rejoice and be merry — and drink to the triumph of science!!

It’s not unbounded joy, of course, because global cancer incidence continues to rise and a number of cancers (e.g., lung, liver and pancreas) remain refractive to all approaches thus far with survival rates stuck below 20%.

A Winter’s Tale

But what’s this? A further, wintry blast of reality from The British Medical Journal no less. It comes from Courtney Davis and her friends at King’s College London and the London School of Economics and Political Science (LSE) who looked at the track record of cancer drugs approved by the EMA between 2009 and 2013. Over this period the EMA approved the use of 48 new cancer drugs.

Charge your glass

It might be a good idea to sit down with a stiff drink at this point and remind ourselves that there are only two aims for cancer drugs: they must either extend the life of the patient or improve their quality of life.

What Dr. D & chums found was — and here, to be absolutely clear, we should quote exactly what they said — “… that most drugs entered the market without evidence of benefit on survival or quality of life. At a minimum of 3.3 years after market entry, there was still no conclusive evidence that these drugs either extended or improved life for most cancer indications. When there were survival gains over existing treatment options or placebo, they were often marginal.”

To be precise, it was 57% (39 of the 68 drugs) that entered the market with no evidence that they improved survival or quality of life.

Cripes!

What does this mean – and how can it be?

Well, first up, clearly a lot of money has been spent by drug companies and health services for absolutely no benefit to patients. Unsurprisingly the authors of the study called on the EMA to “increase the evidence bar for the market authorisation of new cancer drugs.” Which I take to mean ‘get some meaningful data before you stick stuff out there.’ But here’s where things get tricky. If your aim is to extend life, how can you prove a drug works other than by giving it to a significant number of patients and waiting a long time to see what happens?

The way round this has been for clinical trials to use indirect or “surrogate” measures of drug efficacy. The idea is that these endpoints show whether a drug has biological activity and thus might be of clinical use. However, they are not reliable measures of improved quality of life or survival.

So this report leaves us with a long-standing problem. On the one hand there is the understandable drive to get new drugs to patients asap but, on the other, there is the fact that only human beings can model how well a drug works in us. However good your in vitro systems may be and however closely mice may resemble men, they’re not the real thing.

One thing we could do that the report suggests, is to integrate the development and commercialization of cancer drugs at least across the two biggest markets of America and Europe so that the FDA and the EMA don’t appear to be operating in parallel worlds.

All told then, perhaps we should supplant our earlier merriment with the chilling thought that, even after so many years of perspiration and inspiration, cancers still present an immense challenge.

References

Davis, C. et al. (2017). Availability of evidence of benefits on overall survival and quality of life of cancer drugs approved by European Medicines Agency: retrospective cohort study of drug approvals 2009-13. BMJ 2017;359:j4530 doi: 10.1136/bmj.j4530 (Published 2017 October 03).

SEER Cancer Statistics Review (CSR) 1975-2014, updated June 28, 2017.

Cristol, H. (2016). Evolution and Future of Cancer Treatments, The American Cancer Society.

 

Invisible Army Rouses Home Guard

Writing this blog – perhaps any blog – is an odd pastime because you never really know who, if anyone, reads it or what they get out of it. Regardless of that, one person that it certainly helps is me. That is, trying to make sense of the latest cancer news is one of the best possible exercises for making you think clearly – well, as clearly as I can manage!

But over the years one other rather comforting thing has emerged: more and more often I sit down to write a story about a recent bit of science only to remember that it picks up a thread from a piece I wrote months or sometimes years ago. And that’s really cheering because it’s a kind of marker for progression – another small step forward.

Thus it was with this week’s headline news that a ‘cancer vaccine’ might be on the way. In fact this development takes up more than one strand because it’s about immunotherapy – the latest craze – that we’ve broadly explained in Self Help Part-1Gosh! Wonderful GOSH and Blowing-up Cancer and it uses artificial nanoparticles that we met in Taking a Swiss Army Knife to Cancer.

Arming the troops

What Lena Kranz and her friends from various centres in Germany described is yet another twist on the idea of giving our inbuilt defence – i.e. the immune system – a helping hand to tackle tumours. They made small sacs of lipid called nanoparticles (they’re so small you could get 300 in the width of a human hair), loaded them with bits of RNA and injected them into mice. This invisible army of fatty blobs was swept around the circulatory system whereupon two very surprising things happened. The first was that, with a little bit of fiddling (trying different proportions of lipid and RNA), the nanoparticles were taken up by two types of immune cells, with very little appearing in any other cells. This rather fortuitous result is really important because it means that the therapeutic agent (nanoparticles) don’t need to be directly targetted to a tumour cell – thus avoiding one of the perpetual problems of therapy.

The second event that was not at all a ‘gimme’ was that the immune cells (dendritic cells and macrophages) were stimulated to make interferon and they also used the RNA from the nanoparticles as if it was their own to make the encoded proteins – a set of tumour antigens (tumour antigens are proteins made by tumour cells that can be useful in identifying the cells. A large number of have now been found: one group of tumour antigens includes HER2 that we met as a drug target in Where’s That Tumour?)

The interferon was released into the tumour environment in two waves, bringing about the ‘priming’ of T lymphocytes so that, interacting via tumour antigens, they can kill target cells. By contrast with taking cells from the host and carrying out genetic engineering in the lab (Gosh! Wonderful GOSH), this approach is a sort of internal re-wiring achieved by giving a sub-set of immune system cells a bit of genetic code (in the form of RNA).

TAgs RNA Nano picNanoparticle cancer vaccine. Tiny particles (made of lipids) carry RNA into cells of the immune system (dendritic cells and macrophages) in mice. A sub-set of these cells releases a chemical signal (interferon) that promotes the activation of T lymphocytes. The imported RNA is translated into proteins (tumour antigens) – that are presented to T cells. A second wave of interferon (released from macrophages) completes T cell priming so that they are able to attack tumour cells by recognizing antigens on their surface (Kranz et al. 2016; De Vries and Figdor, 2016).

So far Kranz et al. have only tried this method in three patients with melanoma. All three made interferon and developed strong T-cell responses. As with all other immunotherapies, therefore, it is early days but the fact that widely differing strategies give a strong boost to the immune system is hugely encouraging.

Other ‘cancer vaccines’

As a footnote we might add that there are several ‘cancer vaccines’ approved by the US Food and Drug Administration (FDA). These include vaccines against hepatitis B virus and human papillomavirus, along with sipuleucel-T (for the treatment of prostate cancer), and the first oncolytic virus therapy, talimogene laherparepvec (T-VEC, or Imlygic®) for the treatment of some patients with metastatic melanoma.

How was it for you?

As we began by pointing out how good writing these pieces to clarify science is for me, the question for those dear readers who’ve made it to the end is: ‘How did I do?’

References

Kranz, L.M. et al. (2016). Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature (2016) doi:10.1038/nature18300.

De Vries, J. and Figdor, C. (2016). Immunotherapy: Cancer vaccine triggers antiviral-type defences.Nature (2016) doi:10.1038/nature18443.

 

Guess Who’s Coming to Dinner?

 

Question: when is a gene not a gene? Answer: when it’s a pseudogene.

Genes are familiar enough these days when the acronym DNA has become part of everyday speech “It is in Toyota’s DNA that mistakes made once will not be repeated”, as the CEO of Toyota rather sinisterly remarked. You could say that’s pseudo-scientific rubbish but, despite that kind of liberty-taking, most will know that a gene is a stretch of our genetic material (DNA) that carries the code to make a closely related RNA molecule that, in turn, may be used as a template to make a protein ­– it’s the molecular unit of heredity. Well known too is that the Greeks gave us ‘pseudo’ – but what’s a ‘lying’ or ‘false’ gene – and who cares?

No prizes for guessing that we should all be interested because it’s emerging that pseudogenes can be important players in cancer.

Player’s biography

Pseudogenes are somewhat disreputable because they are relatives of normal genes that along the evolutionary highway have become dysfunctional by losing the capacity to be ‘expressed’ – that is, their code can no longer be transformed into RNA and protein. You could think of them as an example of the shambolic way in which species have evolved by random happenstance so that they work in their own particular niches. And if you want the outstanding example of unintelligent design, look no further than yourself, as we did in Holiday Reading (2), Poking the Blancmange.

Just for background, although it doesn’t affect the main story, there are three ways in which our genome can acquire a pseudogene:

1. A normal gene becomes functionally extinct: odd mutational events disable the stretches of DNA that control its expression. The gene is like a siding on a railway that isn’t used for years and years until eventually the points  seize up (it would be a ‘switch’ on US railroads) and the cell machinery can no longer get at it – but when this does happen we get by without that gene.

2. During evolution genes quite often get duplicated – giving multiple copies: if one of these loses its regulatory bits the duplicate gene is switched off – it’s become a ghost.

3. We owe about 8% of our genome to viruses – mainly those with RNA genomes (retroviruses) whose life-cycle turns their RNA into DNA that has then been stuck into our genome. And that’s a lot (about 100,000 bits of retrovirus DNA) especially bearing in mind that only about 1% of our genome encodes proteins.

So our precious genome is littered with corpses and fragments thereof. In the past there’s been a regrettable tendency to label this material as ‘junk’ but increasingly we’re now discovering that there may be genetic life after death, so to speak. It’s not surprising if you think about it. If random events can inactivate a gene then they might do the reverse, even if that may be a much rarer event. And indeed it’s now clear that pseudogenes can be brought back to life through the random mutational events that characterise the rough and tumble of cellular life.

So not all pseudogenes are extinct then?

Correct. Obviously we wouldn’t be wittering on about them had not some bright sparks just shown that pseudogenes – or at least one in particular – can be re-awakened to play a part in cancer. The luminaries are Florian Karreth, Pier Paolo Pandolfi and friends from all over the place (USA, UK, Italy, Singapore) who found that a pseudogene called BRAFP1 (a relative of the normal BRAF gene) can help to drive cancer development. Some earlier studies had shown that BRAFP1 was expressed (i.e. RNA was made from DNA) in various human tumours but Karreth & Co extended this, detecting significant levels of the pseudogene RNA in lymphomas and thyroid tumours and also in cells from melanoma, prostate cancer and lung cancer, whilst it’s not switched on in the corresponding normal cells.

To show that this pseudogene can drive cancers they genetically engineered its over-expression in mice, whereupon the animals developed an aggressive malignancy akin to human lymphoma (specifically diffuse large B cell lymphoma). Short-circuiting an enormous amount of work, it emerged that the pseudogene up-regulated a signaling pathway involving its normal counterpart, BRAF, that drives proliferation.

106 pic

How a pseudogene (BRAFP1) might drive cancer. Top: The scheme illustrates the ‘central dogma’ of molecular biology: DNA makes RNA makes protein. In normal cells a family of micro RNAs (different coloured wiggles) regulate the level of BRAF RNA and hence of BRAF protein (above white line).  Bottom: When the pseudogene BRAFP1 is switched on its RNA competes for the negative regulators: the result is more BRAF RNA making more BRAF protein – making cancer (Karreth et al., 2015).

Interfering RNA

The pseudogene’s RNA manages to interfere with normal control by targeting another type of RNA – micro RNAs, so called because they’re very short (about 20 bases (units) long – so they’re encoded by tiny stretches of the over 3,000 million units that make up the genome). Small they may be but there are hundreds of them and it’s become clear over the last few years that they play critical roles in regulating how much protein is made from specific RNAs. Their method is simple: they recognize (i.e. bind to) stretches of RNA that encode proteins, thereby blocking translation into protein.

Karreth & Co showed that there are about 40 different micro RNAs that can stick to the RNAs encoding BRAF or BRAFP1. Normally when there’s no (or very little) BRAFP1 around they have only BRAF to act on – and their role is to control the proliferation signal it transmits – i.e. to keep that signal to what’s required for normal cell growth control. BUT, when the pseudogene RNA is made in significant amounts the attentions of the 40 micro RNAs are divided. Result: more BRAF RNA, more BRAF protein, higher cell proliferation.

It’s a bit like you’re just sitting down to a family dinner for four when there’s a knock on the door and in walks long lost Uncle Bert, complete with wife and two kids in tow. Of course you invite them to dine too – but now a meal for four has to stretch to eight. There is something for everybody – just not as much. Similarly for the regulators of BRAF: when BRAFP1 is present there’s half as much of the RNA regulators for each – and the result, bearing mind that they are negative regulators, is that the activity of BRAF goes up and the cells proliferate more avidly. The pseudogene is driving cancer.

First but not last

For decades pseudogenes were thought of as ‘junk’ DNA along with most of the rest of the genome that didn’t encode proteins – though I might say that was a concept I never promoted. Beware labeling anything in our genome as junk for it may rise, Kraken like, to remind us of our ignorance. And, now that one pseudogene has come in from the cold and been shown to drive some cancers, you can be confident that others will follow.

References

Karreth, F.A. et al. (2015). The BRAF Pseudogene Functions as a Competitive Endogenous RNA and Induces Lymphoma In Vivo. Cell 161, 319–332.

Dennis’s Pet Menace

As it happened, I’d already agreed to appear on Jeremy Sallis’ Lunchtime Live Show on BBC Radio Cambridgeshire – the plan being just to chat about cancery topics that might be of interest to listeners. Which would have been fine – if only The World Health Organization had left us in peace. But of course they chose last Tuesday to publish their lengthy cogitations on the subject of whether meat is bad for us – i.e. causes cancer.

Cue Press extremism: prime example The Times, quite predictably – they really aren’t great on biomedical science – who chucked kerosene on the barbie with the headline ‘Processed meats blamed for thousands of cancer deaths a year’.

But – to precise facts – and strictly it’s The International Agency for Research on Cancer, the cancer agency of the World Health Organization (WHO), that has ‘evaluated the carcinogenicity of the consumption of red meat and processed meat.’

But hang on … haven’t we been here before?

Indeed we have. As long ago as January 2012 in these pages we commented on the evidence that processed meat can cause pancreatic cancer and in May of the same year we reviewed the cogitations of the Harvard School of Public Health’s 28 year study of 120,000 people that concluded eating red meat contributes to cardiovascular disease, cancer and diabetes. To be fair, that history partially reflects why the WHO Working Group of 22 experts from 10 countries have taken so long to go public: they reviewed no fewer than 800 epidemiological studies! However, as the most frequent target for study was colorectal (bowel) cancer, that was the focus of their report released on 26th October 2015.

So what are we talking about?

Red meat, which means any unprocessed mammalian muscle meat, e.g., beef, veal, pork, lamb, mutton, horse or goat meat, that we usually cook before eating.

Processed meat: any meat not eaten fresh that has been salted, cured, smoked or whatever and commonly treated with chemicals to enhance flavour and colour and to prevent the growth of bacteria.

What did they say?

Processed meat is now classified as carcinogenic to humans – that is it goes into the top group (Group 1) of agents that cause cancer.

Red meat is probably carcinogenic to humans (Group 2A). Group 2B is for things that are possibly carcinogenic to humans.

Why?

Because 12 of the 18 studies they reviewed showed a link between consumption of processed meat and bowel cancer and because it’s known that agents commonly added to processed meat (nitrates and nitrites) can, when we eat them, turn into chemicals that can directly damage DNA, i.e. cause mutations and hence promote cancers.

For red meat 7 out of 15 studies showed positive associations of high versus low consumption with bowel cancer and there is strong mechanistic evidence for a carcinogenic effect i.e. when meat is cooked genotoxic (i.e. DNA-damaging) chemicals can be generated. They put red meat in the probably group because several of the studies that the Working Group couldn’t fault – and therefore couldn’t leave out – showed no association.

Stop woffling

My laptop likes to turn ‘woffling’ into ‘wolfing’. Maybe it’s trying to tell me something.

But is The WHO trying to tell us something specific about wolfing? To be fair, they have a go by estimating that every 50 gram portion of processed meat (say a couple of slices of bacon) eaten daily increases the risk of bowel cancer by about 18%. For red meat the data ‘suggest’ that the risk of bowel cancer could increase by 17% for every 100 gram portion eaten daily.

And what might that mean?

In the UK about 6 people in 100 get bowel cancer: if you take The WHO maximum estimate and have everyone eat 50 grams of processed meat every day of their lives such that 18% more of them would get bowel cancer, the upshot would be 7 people in 100 rather than 6. So it’s a small rise in a relatively small risk.

As the report points out, the Global Burden of Disease Project reckons diets high in processed meat cause about 34,000 cancer deaths per year worldwide and, if the reported associations hold up, the figure for red meat would be 50,000. Compare those figures with smoking that increases the risk of lung cancer by 20-fold and The WHO’s estimate of up to 6 million cancer deaths per year globally caused by tobacco use and 600,000 per year by alcohol consumption.

All of which suggests that it isn’t very helpful to lump meat eating, tobacco and asbestos in the same cancer-causing category and that The WHO could do worse than come up with a new classification system.

And the message?

Unchanged. Remember mankind evolved into the most successful species on the planet as a meat eater. As the advert used to say: It looks good, it tastes good and by golly it does you good – not least as a source of protein, vitamins and other nutrients. Do some exercise and eat a balanced diet – just in case you’ve forgotten, that means limit the amount of red meat (The WHO suggests no more than 30 grams a day for men, 25 g for women) so try fish, poultry, etc. Stick with the ‘good carbs’ (vegetables, fruits, whole grains, etc.), cut out the ‘bad’ (sugar – see Biting the Bitter Bullet), eat fishy fats not saturated fats and, to end on a technical note, don’t pig out.

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‘The Divine Swine’ Castelnuovo Rangone, Italy

Meanwhile back on the Beeb

When the meat story broke I was a bit concerned that we might end up spending the whole of Lunchtime Live on how many bangers are lethal – especially as we were taking calls from listeners. Just in case things became a bit myopic I had Rasher up my sleeve. Rasher, you may recall, was Dennis the Menace‘s pet pig (in the The Beano‘s comic strip) who had a brother (Hamlet), a sister (Virginia Ham) and various other porky rellos. To bring it up to date we’d have introduced Sam Salami and Frank Furter and, of course, Rasher’s grandfather who was the model for the bronze statue named ‘The Divine Swine’ to be found in the little town of Castelnuovo Rangone in Pig Valley, Italy, the home of Parma ham.

But I shouldn’t have worried. All was well in the hands of Jeremy Sallis who, being a brilliant host, ensured that we mainly chatted about meatier matters than what to have for breakfast.

References

Press release: IARC Monographs evaluate consumption of red meat and processed meat.

Q&A on the carcinogenicity of the consumption of red meat and processed meat.

Carcinogenicity of consumption of red and processed meat. www.thelancet.com/oncology Published online October 26, 2015

Wonder of the World

Welcome back from our holidays on which, we trust, you had as much fun reading the four refresher pieces as I had writing them. Utter nonsense, of course. I’ve never found writing to be an orgasmic activity but, as they say about cod liver oil, it is good for you. However, whilst we were all improving ourselves on our deck-chairs and sun-loungers, the Tide of Science was waiting for no man: the waves of cancer biology have obliterated our sand castles and are fast approaching our toes. So let’s get on – albeit doing our best to make the segue from vacation to vocation as seamless as possible …..

So, on the subject of holidays, newspapers and magazines rather like the theme of ‘places to visit before you die’ – which is OK in that the world is wonderful and we should appreciate it. But there’s a problem in that one of the modern wonders is being able to see magnificent photos and movies of every far-flung nook, cranny and creature without leaving our sofa. So when we finally do get off our rear ends and chug past the Statue of Liberty on the Staten Island Ferry, zoom into Sydney or rock up to the Taj Mahal, the reaction is likely to be ‘That’s nice: looks just like on tv. Where next?’

Fortunately, being blasé has its limits. The only time I’ve made it to the Grand Canyon the mid-winter sun highlighted the colours of the rock striations so they were breathtaking in a way no photograph could quite capture. In the same vein, everyone should take the Trans-Siberian Railway we’re often told. And so you should but not because you will see houses and churches, rivers and trees that you can’t find on the Internet but because only borne by the train do you begin to sense the immensity of Mother Russia. The fact that the scenery is almost entirely birch trees minimizes distraction: all you can do is contemplate vastness – and the harshness that brings – an unvarying obbligato to Russian life.

A Provodnitsa looking after one of her passengers on The Trans-Siberian Railway

A Provodnitsa looking after one of her passengers on The Trans-Siberian Railway

The thrice-weekly freight at Grand Canyon Station, circa 1970

The thrice-weekly freight at Grand Canyon Station, circa 1970

 

 

 

 

 

 

Not Forgetting

All of which brings us to something else that is also truly a wonder of the world – cancer. If it seems a trifle weird to describe thus what’s usually classed as one of man’s greatest blights, consider this. The drive to control cancer has generated research on a scale unmatched in any other field of science. One upshot, not necessarily at the top of the list, is that we now have a breathtakingly detailed picture of the astonishing adaptability of life  – that is of our genetic material, DNA, and how its calisthenics can promote the most incredible behaviour on the part of individual cells. It’s true, you might point out, that we can see this by simply looking at the living world around us. The power of DNA to carry, in effect, limitless information produces the infinite cellular variety underpinning the staggering range of life that has evolved on earth. {Did you spot just the other day that a school field trip discovered 13 new species of spider in Queensland – yes, thirteen – inevitably headlined by The Sun as Creepy Hauly}

In the new world

But in focusing on cancers – what happens at the molecular level as they develop and how they evade our attempts to control them – the fine detail of this nigh-on incomprehensible power has been revealed as in no other way.

You’ll know what’s coming: the biggest single boost to this unveiling has been the arrival in the twenty-first century of methods for sequencing DNA and identifying which genes are expressed in cells at any given time. I know: in umpteen blogs I’ve gone on about its awe-inspiring power – but it is stunning and we’re at that stage when new developments leave one gasping almost on a monthly basis. The point here is that it’s not that the science keeps getting turned on its head. Far from it: the message remains that cells pick up changes to their DNA and, with time, these cumulative effects may drive them to make more of themselves than they should.

That’s cancer. But what is fantastic is the molecular detail that the ’omics revolution continues to lay bare. And that’s important because, as we have come to recognize that every cancer is unique, ideally we need to provide specifically tailored treatments, and we can only think of doing that when we know all the facts – even if taking them in demands a good deal of lying down in darkened rooms!

You could think of the fine molecular detail of cancers as corresponding to musical ornaments – flourishes that don’t change the overall tune but without which the piece would be unrecognizable. These include trills and turns – and all musicians will know their appoggiaturas from their acciaccaturas. They’re tiny embellishments – but just try removing them from almost any piece of music.

Lapping at your toes

So let’s look at three recent papers that have used these fabulous methods to unveil as never before the life history of cancers. The first is another masterful offering from The Sanger Institute on breast cancer: an in-depth analysis of 12 patients in which each tumor was sampled from 8 different locations. In the main the mutation patterns differed between regions of the same tumour. They extended this by looking at samples from four patients with multi-focal disease (‘foci’ being small clumps of tumour cells). As expected, individual foci turned out to be clearly genetically related to their neighbours but they also had many ‘private mutations’ – a term usually meaning a mutation found only in a single family or a small population. Here the ‘family’ are individual foci that must have arisen from a common ancestor, and you could think of them as a cellular diaspora – a localised spreading – which makes them a kind of metastasis. Quite often the mutations acquired in these focal sub-clones included major ‘driver’ genes (e.g., P53, PIK3CA and BRCA2). In general such potent mutations tend to be early events but in these foci they’ve appeared relatively late in tumour development. This doesn’t upend our basic picture: it’s just another example of ‘anything goes’ in cancer – but it does make the point that identifying therapeutic targets requires high-depth sequencing to track how individual cancers have evolved through continual acquisition of new mutations and the expansion of individual clones.

The authors used ‘coxcomb’ plots to portray these goings-on but they are quite tricky to make head or tail of. So, to avoid detail overload, I’ve converted some into genetic wallpaper, the non-repeating patterns illustrating the breathtaking variety that has evolved.

Wallpaper jpegDecorative DNA. The discs are ‘coxcomb’ plots – a variant of a pie chart. Here the colours and the wedge sizes represent mutations in different regions of four primary breast tumours. Every disc is different so that the message from this genetic wallpaper is of mutational variation not only between cancers but across the different samples taken from a single tumour. I trust that Lucy Yates, Peter Campbell and their colleagues will not be too upset at my turning their work into art (and greatly abbreviating the story): you can read the original in all its wondrous glory in Nature Medicine 21, 751–759.

The first person to come up with this very graphic way of conveying information was Florence Nightingale who, whilst working in Turkey during the Crimean War, realized that soldiers were dying in the hospitals not only from their wounds but, in much greater numbers, from preventable causes including infections, malnutrition and poor sanitation. Her meticulous recording and original presentation of hospital death tolls made her a pioneer in applied statistics and established the importance of sanitation in hospitals.

Something for the gentlemen

Two equally powerful onslaughts from Gunes Gundem, Peter Campbell and their colleagues at The Sanger Institute (again!) and Dan Robinson and pals from the University of Michigan Medical School have revealed the corresponding molecular detail of prostate cancer. Here too the picture is of each region of a tumour being unique in DNA terms. Moreover, they showed that metastasis-to-metastasis spread was common, either through the seeding of single clones or by the transfer of multiple tumour clones between metastatic sites.

Even that miserable old sod Lenin might have brightened at such fabulous science, before reverting to Eeyore mode with the inevitable “What’s to be done?” But it’s a good question. For example, as a general strategy should we try to kill the bulk of the tumour cells or aim for clones that, although small, carry very potent mutations.

Aside from the basic science, there is one quite bright ray of sunshine: about 90% of the mutations linked with the spread of prostate cancer are potentially treatable with existing drugs. And that really is encouraging, given that the disease kills 11,000 in the UK and over 30,000 in the USA every year.

prostate dogWe might also be heartened by the skills of German Shepherd dogs that can, apparently, be persuaded to apply one of their favourite pastimes – sniffing – to the detection of prostate cancer. Point them at a urine sample and 90% of the time they come up with the right answer. Given the well-known unreliability of the prostate-specific antigen blood test for prostate cancer, it’s nice to think that man’s best friend is on the job.

References

Yates, L.R., et al. (2015). Subclonal diversification of primary breast cancer revealed by multiregion sequencing. Nature Medicine 21, 751–759.

Robinson, D., et al. (2015). Integrative Clinical Genomics of Advanced Prostate Cancer. Cell 161, 1215–1228.

Gundem, G., et al. (2015). The evolutionary history of lethal metastatic prostate cancer. ICGC Prostate UK Group (2015). Nature 520, 353–357.

Holiday Reading (4) – Can We Make Resistance Futile?

For those with a fondness for happy endings we should note that, despite the shortcomings of available drugs, the prospects for patients with a range of cancers have increased significantly over the latter part of the twentieth century. The overall 5-year survival rate for white Americans diagnosed between 1996 and 2004 with breast cancer was 91%; for prostate cancer and non-Hodgkin’s lymphoma the figures were 99% and 66%, respectively. These figures are part of a long-term trend of increasingly effective cancer treatment and there is no doubt that the advances in chemotherapy summarised in the earlier Holiday Readings are contributory factor. Nonetheless, the precise contribution of drug treatments remains controversial and impossible to disentangle quantitatively from other significant factors, notably earlier detection and improved surgical and radiotherapeutic methods.

Peering into the future there is no question that the gradual introduction of new anti-cancer drugs will continue and that those coming into use will be more specific and therefore less unpleasant to use. By developing combinations of drugs that can simultaneously poke the blancmange at several points it may be possible to confront tumor cells with a multiple challenge that even their nimbleness can’t evade, thereby eliminating the problem of drug resistance. Perhaps, therefore, in 20 years time we will have a drug cabinet sufficiently well stocked with cocktails that the major cancers can be tackled at key stages in their evolution, as defined by their genetic signature.

However, on the cautionary side we should note that in the limited number of studies thus far the effect of drug combinations on remission times has not been startling, being measured in months rather than years or decades. Having noted the durability of cancer cells we should not be surprised by this and the concern, of course, is that, however ingenious our efforts to develop drug cocktails, we may always come second to the adaptability of nature.

Equally perturbing is the fact that over 90% of cancer deaths arise from primary tumors spreading to other sites around the body. For this phenomenon, called metastasis, there are currently very few treatment options available and the magnitude of this problem is reflected in the fact that for metastatic breast cancer there has been little change in the survival rates over the past forty years.

Metastasis therefore remains one of the key cancer challenges. It’s over 125 years since the London physician Stepen Paget asked the critical question: ‘What is it that allows tumour cells to spread around the body?’ and it’s a daunting fact that only very recently have we made much progress towards an answer – and thus perhaps a way of controlling it. To the fore in this pursuit has been David Lyden and his colleagues at Weill Cornell Medical College in New York. Their most astonishing finding is that cells in the primary tumour release messengers into the circulation and these, in effect, tag what will become landing points for wandering cells. Astonishing because it means that these sites are determined before any tumour cells actually set foot outside the confines of the primary tumour. Lyden has christened this ‘bookmarking’ cancer. That is a quite remarkable finding – but, as ever in science, it merely shifts the question to ‘OK but what’s the messenger?’

A ray of sunshine

It might appear somewhat churlish, especially after all that funding, to end on a note of defeatist gloom so let’s finish with my ray of sunshine that represents a radical approach to the problem. It relies on the fact that small numbers of cells break away from tumors and pass into the circulation. In addition, tumours can release both DNA and small sacs – like little cells – that contain DNA, proteins and RNAs (nucleic acids closely related to DNA). These small, secreted vesicles are called exosomes – a special form of messengers, communicating with other cells by fusing to them. By transferring molecules between cells, exosomes may play a role in mediating the immune response and they are now recognized as key regulators of tumour growth and metastasis.

Step forward Lyden and friends once more who have just shown in a mouse model of pancreatic cancer that exosomes found their way to the liver during the tumour’s earliest stages. Exosomes are taken up by some of the liver cells and this sets off a chain of cell-to-cell signals that eventually cause the accumulation of a kind of molecular glue (fibronectin). This is the critical ingredient in a microenvironment that attracts tumour cells and promotes their growth as a metastasis (secondary growth). So you can think of exosomes as a kind of environmental educator.

Exosome Fig

Exosomes released from primary tumours can mark a niche for metastasis.

The small sacs of goodies called exosomes are carried to the liver where they fuse with some cells, setting off a chain reaction that produces a sticky protein – fibronectin – a kind of glue for immune cells and tumour cells. (from Costa-Silva, B., Lyden, D. et al., Nature Cell Biology 17, 816–826, 2015).

The recent, remarkable technical advances that permit the isolation of exosomes also make it possible to fish out circulating tumour cells and tumour DNA from a mere teaspoonful of blood.

Circulating tumour cells have already been used to monitor patient responses to chemotherapy – when a treatment works the numbers drop: a gradual rise is the earliest indicator of the treatment failing. Even more exciting, this approach offers the possibility of detecting the presence of cancers years, perhaps decades, earlier than can presently be achieved. Coupling this to the capacity to sequence the DNA of the isolated cells to yield a genetic signature of the individual tumor can provide the basis for drug treatment. There are still considerable reservations attached to this approach but if it does drastically shift the stage at which we can detect tumors it may also transpire that their more naïve forms, in which fewer mutations have accumulated, are more susceptible to inhibitory drugs. If that were to be the case then even our currently rather bare, though slowly expanding, drug cabinet may turn out to be quite powerful.