What’s New in Breast Cancers?

 

One of the best-known things about cancer is that it’s good to catch it early. By that, of course, we don’t mean that you should make an effort to get cancer when you’re young but that, if it does arise it’s a good idea to find out before the initial growth has spread to other places in the body. That’s because surgery and drug treatments are very effective at dealing with ‘primary’ tumours — so much so that over 90% of cancer deaths are caused by cells wandering away from primaries to form secondary growths — a process called metastasis — that are very difficult to treat.

The importance of tumour spreading is shown by the figures for 5-year survival rates. Overall in the USA it’s 90% but this figure falls to below 30% for cancers that have metastasized (e.g., to the lungs, liver or bones). For breast cancer the 5-year survival rate is 99% if it is first detected only in the breast (most cases (62%) are diagnosed at this stage). If it’s spread to blood and lymph vessels in the breast the 5-year survival rate is 85%, dropping to 27% if it’s reached distant parts of the body.

What’s the cause of the problem?

The other thing most people know about cancers is that they’re caused by damage to our genetic material — DNA — that is, by mutations. This raises the obvious notion that secondary tumours might be difficult to deal with because they have accumulated extra mutations compared with those in primaries. And indeed, there have been several studies pointing to just that.

Very recently, however, François Bertucci, Fabrice André and their colleagues in various institutes in France, Switzerland and the USA have mapped in detail the critical alterations in DNA that accumulate as different types of breast cancers develop from early tumours to late, metastatic forms. As is the way these days, their paper contains masses of data but the easiest form of the message comes in the shape of ‘violin plots’. These show the spread of results  — in this case the number of mutations per length of DNA.

Metastatic tumours have a bigger mutational load than early tumours. These plots are for one type of breast tumour (HR+/HER2−) and show results for 381 metastases and 501 early tumours. Red dots = median values: these are the “middle” values rather than an average (or mean) and they show a clear upwards shift in burden as early tumours evolve into metastases. From Bertucci et al., 2019.

The violin plots above are for one subtype of breast cancer (HR+/HER2−). Recall that breast tumours are often defined by which of three types of protein can be detected on the surface of the cells: these are ‘receptors’ that have binding sites for the hormones estrogen and progesterone and for human epidermal growth factor. Hence they are denoted as hormone receptors (HRs) and (human) epidermal growth factor receptor-2 (HER2). Thus tumours may have HRs and HER2 (HR+, HER2+) or various receptors may be undetectable. Triple negative breast cancer (TNBC) is an absence of receptors for both estrogen and progesterone and for HER2.

The plots clearly show an increase in mutation load with progression from early to metastatic tumours (on average from 2.4 to 3.8 mutations per megabase of DNA). Looking at individual genes, nine ‘drivers’ emerged that were more frequently mutated in HR+/HER2− metastatic breast cancers (we described ‘driver’ and ‘passenger’ mutations in Taking Aim at Cancer’s Heart).

So what?

For now these findings give us just a little more insight into what goes on at the molecular level to turn a primary into a metastatic tumour. The fact that some of the acquired driver mutations are associated with poor patient survival offers some guidance as to treatment options.

Don’t get carried away

It’s a familiar story in this field: another small advance in piecing together the jigsaw that is cancer. It doesn’t offer any immediate advance in treatment — mainly because most of the nine ‘driver’ genes identified are tumour suppressors — i.e. they normally act as brakes on cell growth. Mutations knock out that activity and at the moment there is no therapeutic method for reversing such mutations. (The other main class of cancer promoters is ‘oncogenes‘ in which mutations cause hyper-activity).

But such steps are important. The young slave girl in Uncle Tom’s Cabin gave us the phrase “grew like Topsy” — meaning unplanned growth. Cancer growth is indeed unplanned and a bit like Topsy but it’s driven by molecular forces and only through untangling these can we begin to design therapies in a rational way.

Reference

Bertucci, F. et al. (2019). Genomic characterization of metastatic breast cancers. Nature 569, 560–564.

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Secret Army: More Manoeuvres Revealed

 

I don’t know about you but I find it difficult to grasp the idea that there are more bugs in my body than there are ‘me’ cells. That is, microorganisms (mostly bacteria) outnumber the aggregate of liver, skin and what-have-you cells. They’re attracted, of course, to the warm, damp surfaces of the cavities in our bodies that are covered by a sticky, mucous membrane, e.g., the mouth, nose and especially the intestines (the gastrointestinal tract).

The story so far

Over the last few years it’s become clear that these co-residents — collectively called the microbiota — are not just free-loaders. They’re critical to our well-being in helping to fight infection by other microrganisms (as we noted in Our Inner Self), they influence our immune system and in the gut they extract the last scraps of nutrients from our diet. So maybe it makes them easier to live with if we keep in mind that we need them every bit as much as they depend on us.

We now know that there are about 2000 different species of bacteria in the human gut (yes, that really is 2,000 different types of bug) and, with all that diversity, it’s not surprising that the total number of genes they carry far exceeds our own complement (by several million to about 20,000). In it’s a small world we noted that obesity causes a switch in the proportions of two major sub-families of bacteria, resulting in a decrease in the number of bug genes. The flip side is that a more diverse bug population (microbiome) is associated with a healthy status. What’s more, shifts of this sort in the microbiota balance can influence cancer development. Even more remarkably, we saw in Hitchhiker Or Driver? That the microbiome may also play a role in the spread of tumours to secondary sites (metastasis).

Time for a deep breath

If all this is going on in the intestines you might well ask “What about the lungs?” — because, and if you didn’t know you might guess, their job of extracting oxygen from the air we inhale means that they are covered with the largest surface area of mucosal tissue in the body. They are literally an open invitation to passing microorganisms — as we all know from the ease with which we pick up infections.

In view of what we know about gut bugs a rather obvious question is “Could the bug community play a role in lung cancer?” It’s a particularly pressing question because not only is lung cancer the major global cause of cancer death but 70% lung cancer patients have bacterial infections and these markedly influence tumour development and patient survival. Tyler Jacks, Chengcheng Jin and colleagues at the Massachusetts Institute of Technology approached this using a mouse model for lung cancer (in which two mutated genes, Kras and P53 drive tumour formation).

In short they found that germ-free mice (or mice treated with antibiotics) were significantly protected from lung cancer in this model system.

How bacteria can drive lung cancer in mice. Left: scheme of a lung with low levels of bacteria and normal levels of immune system cells. Right: increased levels of bacteria accelerate tumour growth by stimulating the release of chemicals from blood cells that in turn activate cells of the immune system to release other effector molecules that promote tumour growth. The mice were genetically altered to promote lung tumour growth (by mutation of the Kras and P53 genes). In more detail the steps are that the bacteria cause macrophages to release interleukins (IL-1 & IL-23) that stick to a sub-set of T cells (γδ T cells): these in turn release factors that drive tumour cell proliferation, including IL-22. From Jin et al. 2019.

As lung tumours grow in this mouse model the total bacterial load increases. This abnormal regulation of the local bug community stimulates white blood cells (T cells present in the lung) to make and release small proteins (cytokines, in particular interleukin 17) that signal to neutrophils and tumour cells to promote growth.

This new finding reveals that cross-talk between the local microbiota and the immune system can drive lung tumour development. The extent of lung tumour growth correlated with the levels of bacteria in the airway but not with those in the intestinal tract — so this is an effect specific to the lung bugs.

Indeed, rather than the players prominent in the intestines (Bs & Fs) that we met in Hitchhiker Or Driver?, the most common members of the lung microbiome are Staphylococcus, Streptococcus and Lactobacillus.

In a final twist Jin & Co. took bacteria from late-stage tumours and inoculated them into the lungs of mice with early tumours that then grew faster.

These experiments have revealed a hitherto unknown role for bacteria in cancer and, of course, the molecular signals identified join the ever-expanding list of potential targets for drug intervention.

References

Jin, C. et al. (2019). Commensal Microbiota Promote Lung Cancer Development via γδ T Cells. Cell 176, 998-1013.e16.

Fantastic Stuff

 

It certainly is for Judy Perkins, a lady from Florida, who is the subject of a research paper published last week in the journal Nature Medicine by Nikolaos Zacharakis, Steven Rosenberg and their colleagues at the National Cancer Institute in Bethesda, Maryland. Having reached a point where she was enduring pain and facing death from metastatic breast cancer, the paper notes that she has undergone “complete durable regression … now ongoing for over 22 months.”  Wow! Hard to even begin to imagine how she must feel — or, for that matter, the team that engineered this outcome.

How was it done?

Well, it’s a very good example of what I do tend to go on about in these pages — namely that science is almost never about ‘ground-breaking breakthroughs’ or ‘Eureka’ moments. It creeps along in tiny steps, sideways, backwards and sometimes even forwards.

You may recall that in Self Help – Part 2, talking about ‘personalized medicine’, we described how in one version of cancer immunotherapy a sample of a patient’s white blood cells (T lymphocytes) is grown in the lab. This is a way of either getting more immune cells that can target the patient’s tumour or of being able to modify the cells by genetic engineering. One approach is to engineer cells to make receptors on their surface that target them to the tumour cell surface. Put these cells back into the patient and, with luck, you get better tumour cell killing.

An extra step (Gosh! Wonderful GOSH) enabled novel genes to be engineered into the white cells.

The Shape of Things to Come? took a further small step when DNA sequencing was used to identify mutations that gave rise to new proteins in tumour cells (called tumour-associated antigens or ‘neoantigens’ — molecular flags on the cell surface that can provoke an immune response – i.e., the host makes antibody proteins that react with (stick to) the antigens). Charlie Swanton and his colleagues from University College London and Cancer Research UK used this method for two samples of lung cancer, growing them in the lab to expand the population and testing how good these tumour-infiltrating cells were at recognizing the abnormal proteins (neo-antigens) on cancer cells.

Now Zacharakis & Friends followed this lead: they sequenced DNA from the tumour tissue to pinpoint the main mutations and screened the immune cells they’d grown in the lab to find which sub-populations were best at attacking the tumour cells. Expand those cells, infuse into the patient and keep your fingers crossed.

Adoptive cell transfer. This is the scheme from Self Help – Part 2 with the extra step (A) of sequencing the breast tumour. Four mutant proteins were found and tumour-infiltrating lymphocytes reactive against these mutant versions were identified, expanded in culture and infused into the patient.

 

What’s next?

The last step with the fingers was important because there’s almost always an element of luck in these things. For example, a patient may not make enough T lymphocytes to obtain an effective inoculum. But, regardless of the limitations, it’s what scientists call ‘proof-of-principle’. If it works once it’ll work again. It’s just a matter of slogging away at the fine details.

For Judy Perkins, of course, it’s about getting on with a life she’d prepared to leave — and perhaps, once in while, glancing in awe at a Nature Medicine paper that does not mention her by name but secures her own little niche in the history of cancer therapy.

References

McGranahan et al. (2016). Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 10.1126/science.aaf490 (2016).

Zacharakis, N. et al. (2018). Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nature Medicine 04 June 2018.

Hitchhiker Or Driver?

 

It’s a little while since we talked about what you might call our hidden self — the vast army of bugs that colonises our nooks and crannies, especially our intestines, and that is essential to our survival.

In Our Inner Self we noted that these little guys outnumber the human cells that make up the body by about ten to one. Actually that estimate has recently been revised — downwards you might be relieved to hear — to about 1.3 bacterial cells per human cell but it doesn’t really matter. They are a major part of what’s called the microbiome — a vast army of microorganisms that call our bodies home but on which we also depend for our very survival.

In our personal army there’s something like 700 different species of bacteria, with thirty or forty making up the majority. We upset them at our peril. Artificial sweeteners, widely used as food additives, can change the proportions of types of gut bacteria. Some antibiotics that kill off bacteria can make mice obese — and they probably do the same to us. Obese humans do indeed have reduced numbers of bugs and obesity itself is associated with increased cancer risk.

In it’s a small world we met two major bacterial sub-families, Bacteroidetes and Firmicutes, and noted that their levels appear to affect the development of liver and bowel cancers. Well, the Bs & Fs are still around you’ll be glad to know but in a recent piece of work the limelight has been taken by another bunch of Fs — a sub-group (i.e. related to the Bs & Fs) called Fusobacterium.

It’s been known for a few years that human colon cancers carry enriched levels of these bugs compared to non-cancerous colon tissues — suggesting, though not proving, that Fusobacteria may be pro-tumorigenic. In the latest, pretty amazing, installment Susan Bullman and colleagues from Harvard, Yale and Barcelona have shown that not merely is Fusobacterium part of the microbiome that colonises human colon cancers but that when these growths spread to distant sites (i.e. metastasise) the little Fs tag along for the ride! 

Bacteria in a primary human bowel tumour.  The arrows show tumour cells infected with Fusobacteria (red dots).

Bacteria in a liver metastasis of the same bowel tumour.  Though more difficult to see, the  red dot (arrow) marks the presence of bacteria from the original tumour. From Bullman et al., 2017.

In other words, when metastasis kicks in it’s not just the tumour cells that escape from the primary site but a whole community of host cells and bugs that sets sail on the high seas of the circulatory system.

But doesn’t that suggest that these bugs might be doing something to help the growth and spread of these tumours? And if so might that suggest that … of course it does and Bullman & Co did the experiment. They tried an antibiotic that kills Fusobacteria (metronidazole) to see if it had any effect on F–carrying tumours. Sure enough it reduced the number of bugs and slowed the growth of human tumour cells in mice.

Growth of human tumour cells in mice. The antibiotic metronidazole slows the growth of these tumour by about 30%. From Bullman et al., 2017.

We’re still a long way from a human therapy but it is quite a startling thought that antibiotics might one day find a place in the cancer drug cabinet.

Reference

Bullman, S. et al. (2017). Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science  358, 1443-1448. DOI: 10.1126/science.aal5240

One More Small Step

 

Back in the nineteenth century a chap called Augustus De Morgan came up with a set of laws that, when explained in English, sound like the lyrics of a Flanders & Swann song. Opaque to non-maths nerds they may be but they helped to build the mathematics of logic, so next time you meet AND / OR gates in electronics, spare him a thought.

In fact Augustus is rare — maybe unique — among mathematicians in that he’s not completely forgotten, for it was he who penned the lines:

Big fleas have little fleas upon their backs to bite ’em,
And little fleas have lesser fleas, and so, 
ad infinitum.

Given that we now know there’s over 2,500 species of fleas ranging in size from tiny to nearly one centimeter long, it may be literally true. But here, for once, the truth doesn’t matter. It’s a silly rhyme but nonsense verse it is not for it could well serve as a motto for biology because it really captures the essential truth of life: the exquisite choreography of living systems by which incomprehensible numbers of interactions come together to make them work.

Human fleas. Don’t worry: you’ll know if you have them.

Unbidden, De Morgan’s ditty came into my head as I was reading the latest research paper from David Lyden’s group, which he very kindly sent me ahead of publication this week. Avid readers will know the name for we have devoted several episodes (Keeping Cancer Catatonic, Scattering the Bad Seed and Holiday Reading (4) – Can We Make Resistance Futile) to the discoveries of his group in tackling one of the key questions in cancer — namely, how do tumour cells find their targets when they spread around the body? Key because it is this process of ‘metastasis’ that causes most (over 90%) of cancer deaths and if we knew how it worked maybe we could block it.

A succinct summary of those already condensed episodes would be: (1) cells in primary tumours release ‘messengers’ into the circulation that ‘tag’ metastatic sites before any cells actually leave the tumour, (2) the messengers that do the site-tagging are small sacs — mini cells — called exosomes, and (3) they find specific addresses by carrying protein labels (integrins) that home in to different organs — we represented that in the form of a tube train map in Lethal ZIP codes that pulled the whole story together.

The next small step

Now what the folks from Weill Cornell Medicine, New York, Sloan Kettering and a host of other places have done is adapt a flow system to look more closely at exosomes.

Separating small bodies. Particles are injected into a flowing liquid (left) and cross flow at right angles through a membrane (bottom) permits separation on the basis of effective size (called asymmetrical flow field-flow fractionation).

They found that a wide variety of tumour cell types secrete two distinct populations of exosomes — small (60-80 nanometres diameter) and large (90-120 nm). What’s more they found a third type of nanoparticle, smaller than exosomes (less than 50 nm) and without a membrane — so it’s a kind of blob of lipids and proteins (a micelle would be a more scientific term) — that they christened exomeres.

Is it real?

A perpetual problem in biology is reproducibility — that is, whether a new finding can be replicated independently by someone else. Or, put more crudely, do I believe this? This is such an important matter that it’s worth a separate blog but for the moment we’re OK because the results in this paper speak for themselves. First, by using electron microscopy, Lyden et al could actually look at what they’d isolated and indeed discerned three distinct nano-populations — which is how they were able to put the size limits on them.

Electron microscopy of (left) the input mixture (pre-fractionation) and separated fractions: exomere, small exosomes and large exosomes released by tumour cells.. Arrows indicate exomeres (red), small exosomes (blue) and large exosomes (green), from Zhang et al. 2018.

But what’s most exciting in terms of the potential of these results is what’s in the packets. Looking at the fats (lipids), proteins and nucleic acids (DNA and RNA) they contained it’s clear that these are three distinct entities — which makes it very likely they have different effects.

Given their previous finding it must have been a great relief when Lyden & Co identified integrin address proteins in the two exosome sub-populations. But what’s really astonishing is the range of proteins born by these little chaps: something like 400 in exomeres, about 1000 in small exosomes and a similar number in the big ones — and the fact that each contained unique sets of proteins. The new guys — exomeres — carry among other proteins, metabolic enzymes so it’s possible that when they deliver their cargo it might be able to change the metabolic profile of its target. That could be important as we know such changes happen in cancer.

It’s a bewildering picture and working out even the basics of what these little guys do and how it influences cancer is, as we say, challenging. But I think I know a good man for the job!

Augustus De Morgan looking down.

Mathematicians have a bit of a tendency to look down on us experimentalists thrashing around in the undergrowth and I suspect that up in the celestial library, as old Augustus De Morgan thumbed through this latest paper, a slight smile might have come over his face and he could have been heard to murmur: “See, I told you.”

References

Zhang, H. et al. (2018). Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nature Cell Biology 20, 332–343. doi:10.1038/s41556-018-0040-4

Desperately SEEKing …

These days few can be unaware that cancers kill one in three of us. That proportion has crept up over time as life expectancy has gone up — cancers are (mainly) diseases of old age. Even so, they plagued the ancients as Egyptian scrolls dating from 1600 BC record and as their mummified bodies bear witness. Understandably, progress in getting to grips with the problem was slow. It took until the nineteenth century before two great French physicians, Laënnec and Récamier, first noted that tumours could spread from their initial site to other locations where they could grow as ‘secondary tumours’. Munich-born Karl Thiersch showed that ‘metastasis’ occurs when cells leave the primary site and spread through the body. That was in 1865 and it gradually led to the realisation that metastasis was a key problem: many tumours could be dealt with by surgery, if carried out before secondary tumours had formed, but once metastasis had taken hold … With this in mind the gifted American surgeon William Halsted applied ever more radical surgery to breast cancers, removing tissues to which these tumors often spread, with the aim of preventing secondary tumour formation.

Early warning systems

Photos of Halsted’s handiwork are too grim to show here but his logic could not be faulted for metastasis remains the cause of over 90% of cancer deaths. Mercifully, rather than removing more and more tissue targets, the emphasis today has shifted to tumour detection. How can they be picked up before they have spread?

To this end several methods have become familiar — X-rays, PET (positron emission tomography, etc) — but, useful though these are in clinical practice, they suffer from being unable to ‘see’ small tumours (less that 1 cm diameter). For early detection something completely different was needed.

The New World

The first full sequence of human DNA (the genome), completed in 2003, opened a new era and, arguably, the burgeoning science of genomics has already made a greater impact on biology than any previous advance.

Tumour detection is a brilliant example for it is now possible to pull tumour cell DNA out of the gemisch that is circulating blood. All you need is a teaspoonful (of blood) and the right bit of kit (silicon chip technology and short bits of artificial DNA as bait) to get your hands on the DNA which can then be sequenced. We described how this ‘liquid biopsy’ can be used to track responses to cancer treatment in a quick and non–invasive way in Seeing the Invisible: A Cancer Early Warning System?

If it’s brilliant why the question mark?

Two problems really: (1) Some cancers have proved difficult to pick up in liquid biopsies and (2) the method didn’t tell you where the tumour was (i.e. in which tissue).

The next step, in 2017, added epigenetics to DNA sequencing. That is, a programme called CancerLocator profiled the chemical tags (methyl groups) attached to DNA in a set of lung, liver and breast tumours. In Cancer GPS? we described this as a big step forward, not least because it detected 80% of early stage cancers.

There’s still a pesky question mark?

Rather than shrugging their shoulders and saying “that’s science for you” Joshua Cohen and colleagues at Johns Hopkins University School of Medicine in Baltimore and a host of others rolled their sleeves up and made another step forward in the shape of CancerSEEK, described in the January 18 (2018) issue of Science.

This added two new tweaks: (1) for DNA sequencing they selected a panel of 16 known ‘cancer genes’ and screened just those for specific mutations and (2) they included proteins in their analysis by measuring the circulating levels of 10 established biomarkers. Of these perhaps the most familiar is cancer antigen 125 (CA-125) which has been used as an indicator of ovarian cancer.

Sensitivity of CancerSEEK by tumour type. Error bars represent 95% confidence intervals (from Cohen et al., 2018).

The figure shows a detection rate of about 70% for eight cancer types in 1005 patients whose tumours had not spread. CancerSEEK performed best for five types (ovary, liver, stomach, pancreas and esophagus) that are difficult to detect early.

Is there still a question mark?

Of course there is! It’s biology — and cancer biology at that. The sensitivity is quite low for some of the cancers and it remains to be seen how high the false positive rate goes in larger populations than 1005 of this preliminary study.

So let’s leave the last cautious word to my colleague Paul Pharoah: “I do not think that this new test has really moved the field of early detection very far forward … It remains a promising, but yet to be proven technology.”

Reference

D. Cohen et al. (2018). Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 10.1126/science.aar3247.

Boldly Going

When you come across a very successful, middle-aged scientist jumping up and down shouting “This is going to be just amazing” you can only conclude that either the pressures of the life scientific have finally got to him and he’s flipped or there is something really remarkable going on. Thus my feeling this week when I noted the behaviour of Greg Hannon who now works at the Cancer Research Institute in Cambridge.

Probing further, it emerged that Hannon, who is collaborating with Xiaowei Zhuang at Harvard University in the ‘other’ Cambridge, has just been awarded a five-year grant of £20 million by the London-based charity Cancer Research UK as part of its Grand Challenge initiative – more than enough to get your jumping genes going.

But it’s the aim of the project rather than its monetary size that is truly astonishing and has almost a feel of science fiction about it. The plan is nothing less than to come up with an interactive virtual-reality map of breast cancers. That is, to reconstruct every cell that makes up a tumour, showing the different types of cell and what they are up to at any given time – meaning that the model will display the expression level of thousands of genes in each cell and the different proteins being made. Staggering.

What’s the point?

The project is driven by the fact that we have gradually come to realize that tumours are a complex mixture of cells (what’s been called the tumour microenvironment) and the signals that these cells send out and receive determine the extent of tumour growth and whether it can spread to other sites in the body (i.e. metastasize).

Where have we got to?

One approach to mapping what’s going on was laid out a couple of years ago by the converging studies of Rahul Satija and colleagues of the Broad Institute of MIT and Harvard and Kaia Achim et al. from labs in Heidelberg, Cambridge and Oxford using zebrafish embryos and worm brains, respectively.

The method has two parts:

  1. The tissue is dissociated into single cells and the power of sequencing is applied to obtain RNA sequences from each cell (revealing which genes are ‘switched on’ in that cell).
  2. The second step visualizes specific RNAs using tagged probes (fluorescently labeled RNAs that enter cells and bind to target RNAs molecules).

In essence a reference map is made by overlaying the intact tissue with a grid and matching a cell to a grid area by comparing expression of a number of ‘landmark’ genes with the fluorescence marker signal.

To do all this they devised a computational package that, using fewer than 100 landmark genes, maps hundreds of sequenced cells to their location in the tissue. In that arty way that scientists have, they named their package after Georges-Pierre Seurat, the French chappie who came up with the idea of painting in small dots of colour (though his weren’t fluorescent).

Cellular pointillism has just taken another step forward with Keren Bahar Halpern, Ido Amit and Shalev Itzkovitz at the Weizmann Institute of Science, Rehovot, Israel producing a cell-by-cell map of mouse liver, complete with RNA sequences from each cell. To be precise they mapped the hexagon-shaped units called lobules that are repeated to make up mammalian liver.

The shapes of things to come

So the next step for Hannon and his colleagues is an interactive map of a human tumour and, if you can’t wait, CLICK HERE to see their mock-up to give you some idea of what’s in store. In this synthetic video tumour cells are green, macrophages are blue and blood vessels are red.

Overwhelming?

So it’s warp factor 9 for Captain Hannon and his crew. It may be that the 3D images of tumours will look a bit the virtual graphics that the astrophysicists fob off on us whilst pretending they have some idea what a star’s doing umpti-zillion light years away. But in fact, rather than boldly going where no man has gone before“, this cellular journey is better summed up by Marcel Proust The real voyage of discovery consists not in seeking new landscapes, but in having new eyes” – the new eyes being the stunning combination of methods that permits 3D interrogation of individual cells.

Will this phase of the Grand Challenge produce overwhelming amounts of data? Undoubtedly. But, if we are to understand how living things work we have to front up to the complexity of nature. We then have to hope we are smart enough to resolve the crucial from the detail.

References

Satija, R. et al. (2015). Spatial reconstruction of single-cell gene expression data. Nature Biotechnology 33, 495–502.

Achim, K. et al. (2015). High-throughput spatial mapping of single-cell RNA-seq data to tissue of origin. Nature Biotechnology 33, 503–509.

Halpern, K. B. et al. (2017). Nature 542, 352–356.

Open Wide for Pasty’s Throat

 

Once upon a time (1903 to be exact) a very rich Adelaide family acquired a new member in the form of a little boy whom they christened Norman. Most of the family were doctors and, so well-heeled were they, when young Norm reached the age of 10 they clubbed together and sent him off to the Old Country – and not just to any bit of Merrie England but to Eton (the school, of course, not the rustic parish, generally held to be the most expensive of all – fees currently about £36,000 a year, not counting extras). Norm never returned: South Australia’s loss was Britain’s gain.

We all know what happens to kids that go to Eton – but our Adelaide man was different. For one thing he was very bright and for another he had his family’s love of medicine. He ended up specializing in the thorax – the bit between the neck and the tummy that includes the oesophagus, commonly known as the foodpipe or gullet. Eton probably helped get him started but, even more usefully, some bright spark there gave him the nickname ‘Pasty’ – so great an improvement on Norman that it stuck for life. ‘Pasty’ Barrett ended up as a consultant at St. Thomas’ Hospital where, in 1947, he successfully repaired a ruptured oesophagus – a surgical first for a hitherto fatal condition.

Shortly after that, in 1950, he described finding that sometimes the cells lining the gullet change in appearance, switching from multiple layers of flat cells to a single layer of cells that look like those found in the intestine. We know now that this change is caused by acid from the stomach being squeezed up into the oesophagus. Occasional regurgitation is called heartburn but when it’s persistent it becomes gastric reflux disease – and in about 10% of those cases sustained irritation caused by the stomach juices upsets the cells lining the gullet and they undergo the change to what is now called Barrett’s oesophagus.

Who cares about Barrett’s?

Well, we should all at least take note because a few percent of those with Barrett’s oesophagus will get cancer of the oesophagus, which is now the sixth most common cause of cancer-related death world-wide. Oesophageal cancer has become more common over the last 40 years, men are more prone to it than women and it kills about 15,000 people in the USA each year and nearly 8,000 in the UK. It’s very bad news. Most cases aren’t discovered until the disease has spread and it is then more or less untreatable. The prognosis is dismal: the five-year survival figure is barely 15%. Part of the problem is that the main sign is pain or difficulty in swallowing, often ignored until it is too late.

For many years the only way of finding abnormal tissue was by an endoscopy – a tube with a camera pushed down the throat – both unpleasant and expensive. There has, therefore, been a desperate need for an easy, cheap, non-invasive test to screen for Barrett’s oesophagus.

Professor Rebecca Fitzgerald

     Professor Rebecca             Fitzgerald

Pill on a string

Enter Rebecca Fitzgerald, a member of the Department of Oncology in Cambridge and a consultant at Addenbrooke’s Hospital, with a brilliantly simple development from earlier attempts to screen the lining of the gullet. The patient swallows a kind of tea-bag on a string which is then pulled up from the stomach. The ‘tea-bag’ is actually a capsule about the size of a multi-vitamin pill containing a sort of honeycomb sponge covered with a coating that dissolves in a few minutes when it reaches the stomach. As the sponge comes up it picks up cells from the gullet lining (about half a million of them) that can then be analysed. The whole gizmo’s called a ‘Cytosponge’. It works with no problems and because it collects cells from the length of the gullet it gives a complete picture, rather than the local regions sampled in biopsies.

Pill on a string

                 Pill on a string

Cytosponge (left) and being drawn up the gullet (right)

       Cytosponge (left) and being             drawn up the gullet (right)

 

 

 

 

 

What we’ve learned

The hope was that the cells picked up by Cytosponge could be sequenced – i.e. their DNA code could be obtained – and that this would reveal the stages of oesophageal cancer development and hence whether a given case of Barrett’s would or would not progress to cancer. The phases of Barrett’s oesophagus involve a change in the shape of cells lining the tube (from thin, flat cells called squamous epithelial cells to taller columnar cells resembling those in the intestine). This change is called metaplasia: the abnormal cells may then proliferate (dysplasia). If this stage can be detected it’s possible to remove the abnormal tissue by using endoscopic therapy before the condition progresses to full carcinoma.

Remarkably, whole-genome sequences from Barrett’s and from oesophageal carcinoma showed that multiple mutations (changes in DNA sequence) accumulate even in cells that are over-proliferating but look normal. The picture is similar to the ‘battlefield of hundreds of competing mutant clones’ in normal eyelid skin that we saw in The Blink of an Eye.

As the condition progresses the range of mutations increases: in particular, regions of DNA are duplicated – so that the genes therein are present in abnormal numbers. Typically there were 12,000 mutations per person with Barrett’s oesophagus without cancer and 18,000 mutations within the cancer.

Even from this mayhem there emerged mutation patterns (changes in the letters of the DNA code, e.g., A to a G or C to a T) characteristic of the damage caused to the cells lining the oesophagus by splashing stomach acid. These ‘fingerprints’ were found in both Barrett’s and oesophageal cancer – consistent with them being very early events – parallelling the specific mutations in lung cancer caused by tobacco carcinogens.

But …

The great hope was that the spectrum of mutations would identify precursors to cancer and hence those patients requiring treatment. In fact these horribly heterogeneous tissues – a real genetic gemisch – show surprisingly little mutational overlap between Barrett’s oesophagus and oesophageal cancer.

However, it’s possible to take the cells collected by the Cytosponge and screen them for the presence of specific proteins (using antibodies) and it turns out that one in particular, TFF3 (Trefoil Factor 3), provides a highly accurate diagnosis of Barrett’s oesophagus. In addition, although the genetic changes that occur during the progression from Barrett’s to cancer are complex, mutations in one gene (P53 – the ‘guardian of the genome’) are common in pre-cancerous, high grade dysplasia and thus provide an indicator of risk.

All of which means that we haven’t ‘conquered’ oesophageal cancer – but thanks to these remarkable advances we have a much better understanding of its molecular basis. Even more importantly, it’s possible to detect the early stages – and do something about it.

AND … whilst making a major contribution to all this, Rebecca Fitzgerald very kindly found time to make suggestions and provide additional information for this piece.

References

Ross-Innes, C.S., Fitzgerald, R.C. et al. (2015). Nature Genetics 47, 1038-1046.

 

Pass the Aspirin

And so you should if you’ve got a headache – unless, of course, you prefer paracetamol. There can scarcely be anyone who hasn’t resorted to a dose of slightly modified salicylic acid (For the chemists: its hydroxyl group is converted into an ester group (R-OH → R-OCOCH3) in aspirin), given that the world gobbles up an estimated 40,000 tonnes of the stuff every year. It’s arguable, therefore, that an obscure clergyman by the name of Edward Stone has done more for human suffering than pretty well anyone, for it was he who, in 1763, made a powder from the bark of willow trees and discovered its wondrous property. The bark and leaves had actually been used for centuries – back at least to the time of Hippocrates – for reducing pain and fever, although it wasn’t until 1899 that Aspirin made its debut on the market and it was 1971 before John Vane discovered how it actually worked. He got a Nobel Prize for showing that it blocks production of things called prostaglandins that act a bit like hormones to regulate inflammation (for the chemists – again! – it irreversibly inactivates the enzyme cyclooxygenase, known as COX to its pals).

Daily pill popping

Aside from fixing the odd ache, over the years evidence has gradually accumulated that people at high risk of heart attack and those who have survived a heart attack should take a low-dose of aspirin every day. In addition to decreasing inflammation (by blocking prostaglandins) aspirin inhibits the formation of blood clots – so helping to prevent heart attack and stroke. Almost as a side-effect the studies that have lead to this being a firm recommendation have also shown that aspirin may reduce the risk of cancers, particularly of the bowel (colorectal cancer). Notably, Peter Rothwell and colleagues from Oxford showed that daily aspirin taken for 10 years reduced the risk of bowel cancer by 24% and also protected against oesophageal cancer – and a more recent analysis has broadly supported these findings. In addition they have also found that aspirin lowers the risk of cancers spreading around the body, i.e. forming distant metastases.

Why is aspirin giving us a headache – again?

First because a large amount of media coverage has been given to a report from Leiden University Medical Center, presented at The European Cancer Congress in September, that used Dutch records to see whether taking aspirin after being diagnosed with gastrointestinal cancer influenced survival. Their conclusion was that patients using aspirin after diagnosis doubled their survival chances compared with those who did not take aspirin. Needless to say, these words have been trumpeted by newspapers from The Times to the Daily Mail in the usual fashion (“Aspirin could almost double your chance of surviving cancer”). Unfortunately we can’t lay all the blame on the press: the authors of the report used the tactic of issuing a Press Release, a thoroughly reprehensible ploy for gaining attention when the work involved has not been peer reviewed. (The point here for non-scientists is that you can stand up at a meeting and say the moon’s made of blue cheese and it’s fine. Only after your work has been assessed by colleagues in the course of the normal publication process does it begin to have some credibility). So there’s a problem here, with what was an ‘observational study’, as to just what the findings mean – and the wise thing is to wait for the results of a ‘randomised controlled trial’ that is under way. 

The second source of mental strain is down to the ferociously named United States Preventive Services Task Force that has just (September 2015) come up with the recommendation that we should take aspirin to prevent bowel cancer. Why should we pay any attention? Because the ‘Force’ are appointed by the US Department of Health and they wield great influence upon medical practice – and because it’s the first time a major American medical organization has issued a broad recommendation to take aspirin to prevent a form of cancer.

In this latest oeuvre they confirm that the well-known risks attached to aspirin-eating (ulcers and stomach bleeding) are out-weighed by the protection against heart disease in those between the ages of 50 and 69 who are at high risk (e.g., have a history of heart attacks). If you feel your heart can take the strain you can find out your risk by using the National Heart, Lung, and Blood Institute’s online risk assessment tool. To get an answer you need to know your age, sex (i.e. gender, as its called these days), cholesterol levels (total and high density lipoproteins, HDLs – they’re the ‘good’ cholesterol), whether you smoke and your systolic blood pressure (that’s the X in X/Y).

This is such a critical issue it’s worth seeing what the Task Force actually said: “The USPSTF recommends low-dose aspirin use for the primary prevention of cardiovascular disease (CVD) and colorectal cancer in adults ages 50 to 59 years who have a 10% or greater 10-year CVD risk, are not at increased risk for bleeding, have a life expectancy of at least 10 years, and are willing to take low-dose aspirin daily for at least 10 years.”

If you’re younger than 50 or over 70 you’re on your own: the Force doesn’t recommend anything. And if you’re 60 to 69 the wording of their advice is wonderfully delicate: The decision to use low-dose aspirin to prevent CVD (cardiovascular disease) and colorectal cancer in adults ages 60 to 69 years who have a greater than 10% 10-year CVD risk should be an individual one.”

So that’s cleared that up …

Er, not quite. Various luminaries have been quick to demur. For example, Dr. Steven Nissen, the chairman of cardiology at the Cleveland Clinic has opined that the Task Force “has gotten it wrong.” In other words aspirin does more harm than good – though he might be a bit late as seemingly an astonishing 40% of Americans over the age of 50 take aspirin to prevent cardiovascular disease. I reckon that’s about 40 million people. Mmm … so that’s where the 40,000 tonnes goes (well, about one-fifth of it).

What’s the advice?

We’re more or less where we came in. I take an aspirin, or more usually a paracetamol, when I’ve got a stonking headache. Otherwise I wouldn’t take any kind of pill or supplement unless there is an overwhelming medical case for so doing. And pill-poppers out there might note the findings of Eva Saedder and her pals at Aarhus University that the single, strongest independent risk factor for drug-induced serious adverse events is the number of drugs that the patient is taking.

References

Rothwell, P. et al. (2012). Short-term effects of daily aspirin on cancer incidence, mortality, and non-vascular death: analysis of the time course of risks and benefits in 51 randomised controlled trials, Lancet DOI:1016/S0140-6736(11)61720-0

Rothwell P. et al. (2012). Effect of daily aspirin on risk of cancer metastasis: a study of incident cancers during randomised controlled trial, Lancet DOI:1016/S0140-6736(12)60209-8

Lancet editorial on Rothwell et al. 2011.

Algra, A. and Rothwell, P. (2012). Effects of regular aspirin on long-term cancer incidence and metastasis: a systematic comparison of evidence from observational studies versus randomised trials, Lancet Oncology DOI:10.1016/S1470-2045(12)70112-2.

Frouws M et al. Aspirin and gastro intestinal malignancies; improved survival not only in colorectal cancer? Conference abstract. European Cancer Congress 2015

Press release: Post diagnosis aspirin improves survival in all gastrointestinal cancers. The European Cancer Congress 2015. September 23 2015

Cuzick J, Thorat MA, Bosetti C, et al. Estimates of benefits and harms of prophylactic use of aspirin in the general population. Annals of Oncology. Published online August 5 2014

U.S. Preventive Services Task Force Draft Recommendation Statement: Aspirin to Prevent Cardiovascular Disease and Cancer

Saedder, E.A. et al. (2015). Number of drugs most frequently found to be independent risk factors for serious adverse reactions: a systematic literature review. British Journal of Clinical Pharmacology 80, 808–817.

 

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