Through the Smokescreen

For many years I was lucky enough to teach in a cancer biology course for third year natural science and medical students. Quite a few of those guys would already be eyeing up research careers and, within just a few months, some might be working on the very topics that came up in lectures. Nothing went down better, therefore, than talking about a nifty new method that had given easy-to-grasp results clearly of direct relevance to cancer.

Three cheers then for Mikhail Denissenko and friends who in 1996 published the first absolutely unequivocal evidence that a chemical in cigarette smoke could directly damage a bit of DNA that provides a major protection against cancer. The compound bound directly to several guanines in the DNA sequence that encodes P53 – the protein often called ‘the guardian of the genome’ – causing mutations. A pity poor old Fritz Lickint wasn’t around for a celebratory drink – it was he, back in the 1930s, that first spotted the link between smoking and lung cancer.

This was absolutely brilliant for showing how proteins switched on genes – and how that switch could be perturbed by mutations – because, just a couple of years earlier, Yunje Cho’s group at the Memorial Sloan-Kettering Cancer Center in New York had made crystals of P53 stuck to DNA and used X-rays to reveal the structure. This showed that six sites (amino acids) in the centre of the P53 protein poked like fingers into the groove of double-stranded DNA.

x-ray-picCentral core of P53 (grey ribbon) binding to the groove in double-stranded DNA (blue). The six amino acids (residues) most commonly mutated in p53 are shown in yellow (from Cho et al., 1994).

So that was how P53 ‘talked’ to DNA to control the expression of specific genes. What could be better then, in a talk on how DNA damage can lead to cancer, than the story of a specific chemical doing nasty things to a gene that encodes perhaps the most revered of anti-cancer proteins?

The only thing baffling the students must have been the tobacco companies insisting, as they continued to do for years, that smoking was good for you.

And twenty-something years on …?

Well, it’s taken a couple of revolutions (scientific, of course!) but in that time we’ve advanced to being able to sequence genomes at a fantastic speed for next to nothing in terms of cost. In that period too more and more data have accumulated showing the pervasive influence of the weed. In particular that not only does it cause cancer in tissues directly exposed to cigarette smoke (lung, oesophagus, larynx, mouth and throat) but it also promotes cancers in places that never see inhaled smoke: kidney, bladder, liver, pancreas, stomach, cervix, colon, rectum and white blood cells (acute myeloid leukemia). However, up until now we’ve had very little idea of what, if anything, these effects have in common in terms of molecular damage.

Applying the power of modern sequencing, Ludmil Alexandrov of the Los Alamos National Lab, along with the Wellcome Trust Sanger Institute’s Michael Stratton and their colleagues have pieced together whole-genome sequences and exome sequences (those are just the DNA that encode proteins – about 1% of the total) of over 5,000 tumours. These covered 17 smoking-associated forms of cancer and permitted comparison of tobacco smokers with never-smokers.

Let’s hear it for consistent science!

The most obvious question then is do the latest results confirm the efforts of Denissenko & Co., now some 20 years old? The latest work found that smoking could increase the mutation load in the form of multiple, distinct ‘mutational signatures’, each contributing to different extents in different cancers. And indeed in lung and larynx tumours they found the guanine-to-thymine base-pair change that Denissenko et al had observed as the result of a specific chemical attaching to DNA.

For lung cancer they concluded that, all told, about 150 mutations accumulate in a given lung cell as a result of smoking a pack of cigarettes a day for a year.

Turning to tissues that are not directly exposed to smoke, things are a bit less clear. In liver and kidney cancers smokers have a bigger load of mutations than non-smokers (as in the lung). However, and somewhat surprisingly, in other smoking-associated cancer types there were no clear differences. And even odder, there was no difference in the methylation of DNA between smokers and non-smokers – that’s the chemical tags that can be added to DNA to tune the process of transforming the genetic code into proteins. Which was strange because we know that such ‘epigenetic’ changes can occur in response to external factors, e.g., diet.

What’s going on?

Not clear beyond the clear fact that tissues directly exposed to smoke accumulate cancer-driving mutations – and the longer the exposure the bigger the burden. For tissues that don’t see smoke its effect must be indirect. A possible way for this to happen would be for smoke to cause mild inflammation that in turn causes chemical signals to be released into the circulation that in turn affect how efficiently cells repair damage to their DNA.

raleighs_first_pipe_in_england-jpeg

Sir Walt showing off on his return                         to England

Whose fault it is anyway?

So tobacco-promoted cancers still retain some of their molecular mystery as well as presenting an appalling and globally growing problem. These days a popular pastime is to find someone else to blame for anything and everything – and in the case of smoking we all know who the front-runner is. But although Sir Walter Raleigh brought tobacco to Europe (in 1578), it had clearly been in use by American natives long before he turned up and, going in the opposite direction (à la Marco Polo), the Chinese had been at it since at least the early 1500s. To its credit, China had an anti-smoking movement by 1639, during the Ming Dynasty. One of their Emperors decreed that tobacco addicts be executed and the Qing Emperor Kangxi went a step further by beheading anyone who even possessed tobacco.

And paying the price

And paying the price

If you’re thinking maybe we should get a touch more Draconian in our anti-smoking measures, it’s worth pointing out that the Chinese model hasn’t worked out too well so far. China’s currently heading for three million cancer deaths annually. About 400,000 of these are from lung cancer and the smoking trends mean this figure will be 700,000 annual deaths by 2020. The global cancer map is a great way to keep up with the stats of both lung cancer and the rest – though it’s not for those of a nervous disposition!

References

Denissenko, M.F. et al. ( (1996). Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53.Science 274, 430–432.

Cho, Y. et al. (1994). Crystal Structure of a p53 Tumor Suppressor-DNA Complex: Understanding Tumorigenic Mutations. Science, 265, 346-355.

Alexandrov, L.D. et al. (2016). Mutational signatures associated with tobacco smoking in human cancer. Science 354, 618-622.

Gosh! Wonderful GOSH

Anyone who reads these pages will long ago, I trust, have been persuaded that the molecular biology of cells is fascinating, beautiful and utterly absorbing – and all that is still true even when something goes wrong and cancers make their unwelcome appearance. Which makes cancer a brilliant topic to talk and write about – you know your audience will be captivated (well, unless you’re utterly hopeless). There’s only one snag, namely that – perhaps because of the unwelcome nature of cancers – it’s tough to make jokes. One of the best reviews I had for Betrayed by Nature was terrifically nice about it but at the end, presumably feeling that he had to balance things up, the reviewer commented that it: “..is perhaps a little too light-hearted at times…” Thank you so much anonymous critic! Crikey! If I’d been trying to do slap-stick I’d have bunged in a few of those lewd chemicals – a touch of erectone, a bit of PORN, etc. (btw, the former is used in traditional Chinese medicine to treat arthritis and the latter is poly-ornithinine, so calm down).

I guess my serious referee may have spotted that I included a poem – well, two actually, one written by the great JBS Haldane in 1964 when he discovered he had bowel cancer which begins:

I wish I had the voice of Homer

To sing of rectal carcinoma,

Which kills a lot more chaps, in fact,
Than were bumped off when Troy was sacked.

Those couplets may reflect much of JBS with whom I can’t compete but, nevertheless, in Betrayed by Nature I took a deep breath and had a go at an update that began:

Long gone are the days of Homer
But not so those of carcinoma,
Of sarcoma and leukemia

And other cancers familia.
But nowadays we meet pre-school
That great and wondrous Molecule.
We know now from the knee of Mater
That DNA’s the great creator.

and went on:

But DNA makes cancer too

Time enough—it’ll happen to you.
“No worries sport” as some would say,
These days it’s “omics” all the way.

So heed the words of JBS

Who years ago, though in distress,
Gave this advice on what to do

When something odd happens to you:
“Take blood and bumps to your physician
Whose only aim is your remission.”

I’d rather forgotten my poem until in just the last week there hit the press a story illustrating that although cancer mayn’t be particularly fertile ground for funnies it does gloriously uplifting like nothing else. It was an account of how science and medicine had come together at Great Ormond Street Hospital to save a life and it was even more thrilling because the life was that of a little girl just two years old. The saga brought my poem to mind and it seemed, though I say it myself, rather spot on.

The little girl, Layla, was three months old when she was diagnosed with acute lymphoblastic leukemia (ALL) caused by a piece of her DNA misbehaving by upping sticks and moving to a new home on another chromosome – one way in which genetic damage can lead to cancer. By her first birthday chemotherapy and a bone marrow transplant had failed and the only remaining option appeared to be palliative care. At this point the GOSH team obtained special dispensation to try a novel immunotherapy using what are being called “designer immune cells“. Over a few months Layla recovered and is now free of cancer. However, there are no reports of Waseem Qasim and his colleagues at GOSH and at University College London dancing and singing the Trafalgar Square fountains – they’re such a reserved lot these scientists and doctors.

How did they do it?

In principle they used the gene therapy approach that, helpfully, we described recently (Self Help Part 2). T cells isolated from a blood sample have novel genes inserted into their DNA and are grown in the lab before infusing into the patient. The idea is to improve the efficiency with which the T cells target a particular protein (CD19) present on the surface of the leukemia cells by giving them artificial T cell receptors (also known as chimeric T cell receptors or chimeric antigen receptors (CARs) – because they’re made by fusing several bits together to make something that sticks to the target ‘antigen’ – CD19). The engineered receptors thereby boost the immune response against the leukemia. The new genetic material is inserted into a virus that carries it into the cells. So established is this method that you can buy such modified cells from the French biotech company Cellectis.

105 picAdoptive cell transfer immunotherapy. T cells are isolated from a blood sample and novel genes inserted into their DNA. The GOSH treatment also uses gene editing by TALENs to delete two genes. The engineered T cells are expanded, selected and then infused into the patient.

Is that all?

Not quite. To give themselves a better chance the team added a couple of extra tricks. First they included in the virus a second gene, RQR8, that encodes two proteins – this helps with identifying and selecting the modified cells. The second ploy is, perhaps, the most exciting of all: they used gene editing – a rapidly developing field that permits DNA in cells to be modified directly: it really amounts to molecular cutting and pasting. Also called ‘genome editing’ or ‘genome editing with engineered nucleases’ (GEEN), this form of genetic engineering removes or inserts sections of DNA, thereby modifying the genome.

The ‘cutting’ is done by proteins (enzymes called nucleases) that snip both strands of DNA – creating double-strand breaks. So nucleases are ‘molecular scissors.’ Once a double-strand break has been made the built-in systems of cells swing into action to repair the damage (i.e. stick the DNA back together as best it can without worrying about any snipped bits – these natural processes are homologous recombination and non-homologous end-joining, though we don’t need to bother about them here).

To be of any use the nucleases need to be targeted – made to home in on a specific site (DNA sequence) – and for this the GOSH group used ‘transcription activator-like effectors’ (TALEs). The origins of these proteins could hardly be further away from cancer – they come from a family of bacteria that attacks hundreds of different types of plants from cotton to fruit and nut trees, giving rise to things like citrus canker and black rot. About six years ago Jens Boch of the Martin-Luther-University in Halle and Adam Bogdanove at Iowa State University with their colleagues showed that these bugs did their dirty deeds by binding to regulatory regions of DNA thereby changing the expression of genes, hence affecting cell behavior. It turned out that their specificity came from a remarkably simple code formed by the amino acids of TALE proteins. From that it’s a relatively simple step to make artificial TALE proteins to target precise stretches of DNA and to couple them to a nuclease to do the cutting. The whole thing makes a TALEN (transcription activator-like effector nuclease). TALE proteins work in pairs (i.e. they bind as dimers on a target DNA site) so an artificial TALEN is like using both your hands to grip a piece of wood either side of the point where, using your third hand, you make the cut. The DNA that encodes the whole thing is inserted into plasmids that are transfected into the target cells; the expressed gene products then enter the nucleus to work on the host cell’s genome. There are currently three other approaches to nuclease engineering (zinc finger nucleases, the CRISPR/Cas system and meganucleases) but we can leave them for another time.

The TALENs made by the GOSH group knocked out the T cell receptor (to eliminate the risk of an immune reaction against the engineered T cells (called graft-versus-host disease) and CD52 (encodes a protein on the surface of mature lymphocytes that is the target of the monoclonal antibody alemtuzumab – so this drug can be used to prevent rejection by the host without affecting the engineered T cells).

What next?

This wonderful result is not a permanent cure for Layla but it appears to be working to stave off the disease whilst she awaits a matched T cell donor. It’s worth noting that a rather similar approach has been used with some success in treating HIV patients but it should be born in mind that, brilliant though these advances are, they are not without risks – for example, it’s possible that the vector (virus) that delivers DNA might have long-term effects – only time will tell.

Almost the most important thing in this story is what the GOSH group didn’t do. They used the TALENs gene editing method to knock out genes but it’s also a way of inserting new DNA. All you need to do is add double-stranded DNA fragments in the correct form at the same time and the cell’s repair system will incorporate them into the genome. That offers the possibility of being able to repair DNA damage that has caused loss of gene function – a major factor in almost all cancers. Although there is still no way of tackling the associated problem of how to target gene editing to tumour cells, it may be that Layla’s triumph is a really significant step for cancer therapy.

Reference

Smith, J. et al. (2015). UCART19, an allogeneic “off-the-shelf” adoptive T-cell immunotherapy against CD19+ B-cell leukemias. Journal of Clinical Oncology 33, 2015 (suppl; abstr 3069).

 

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.

_65259128_6136791400_49fc5aaece_b

‘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

Holiday Reading (3) – Stopping the Juggernaut

The mutations that drive cancers fall into two major groups: those that reduce or eliminate the activity of affected proteins and those that have the opposite effect and render the protein abnormally active. It’s intuitively easy to see how the latter work: if a protein (or more than one) in a pathway that tells cells to proliferate becomes more efficient the process is accelerated. Less obvious is how losing an activity might have a similar effect but this comes about because the process by which one cell becomes two (called the cell cycle) is controlled by both positive and negative factors (accelerators and brakes if you will). This concept of a balancing act – signals pulling in opposite directions – is a common theme in biology. In the complex and ever changing environment of a cell the pressure to reproduce is balanced by cues that ask crucial questions. Are there sufficient nutrients available to support growth? Is the DNA undamaged, i.e. in a fit state to be replicated? If the answer to any of these questions is ‘no’ the cell cycle machinery applies the brakes, so that operations are suspended until circumstances change. The loss of negative regulators releases a critical restraint so that cells have a green light to divide even when they should not – a recipe for cancer.

Blanc sides.004

The cell cycle.

Cells are stimulated by growth factors to leave a quiescent state (G0) and enter the cell cycle – two growth phases (G1 & G2), S phase where DNA is duplicated and mitosis (M) in which the cells divide to give to identical daughter cells. Checkpoints can arrest progression if, for example, DNA is damaged. 

We’re all familiar with this kind of message tug-of-war at the level of the whole animal. We’ve eaten a cream cake and the schoolboy within is saying ‘go on, have another’ whilst the voice of wisdom is whispering ‘if you go on for long enough you’ll end up spherical.’

Because loss of key negative regulators occurs in almost all cancers it is a high priority to find ways of replacing inactivated or lost genes. Thus far, however, successful ‘gene therapy’ approaches have not been forthcoming with perhaps the exception of the emerging field of immunotherapy. The aim here is to boost the activity of the immune system of an individual – in other words to give an innate anti-cancer defense a helping hand. The immune system can affect solid cancers through what’s become known as the tumour microenvironment – the variety of cells and messengers that flock to the site of the abnormal growth. We’ve referred to these as ‘groupies’ and they include white blood cells. They’re drawn to the scene of the crime by chemical signals released by the tumour – the initial aim being to liquidate the intruder (i.e. tumour cells). However, if this fails, a two-way communication sees would-be killers converted to avid supporters that are essential for cancer development and spread.

Blanc sides.002

The tumour microenvironment. Tumour cells release chemical messengers that attract other types of cell, in particular those that mediate the immune response. If the cancer cells are not eliminated a two-way signaling system is established that helps tumour development.

There is much optimism that this will evolve into a really effective therapy but it is too early for unreserved confidence.

The obstacle of reversing mutations that eliminate the function of a gene has led to the current position in which practically all anti-cancer agents in use are inhibitors, that is, they block the activity of a protein (or proteins) resulting in the arrest of cell proliferation – which may ultimately lead to cell death. Almost all these drugs are not specific for tumour cells: they hit some component of the cell replication machinery and will block division in any cell they reach – which is why so many give rise to the side-effects notoriously associated with cancer chemotherapy. For example, the taxanes – widely used in this context – stick to protein cables to prevent them from pulling duplicated DNA strands apart so that cells, in effect, become frozen in final stages of division. Other classes of agent target different aspects of the cell cycle.

It is somewhat surprising that non-tumour specific agents work as well as they do but their obvious shortcomings have provided a major incentive for the development of ‘specific’ drugs – meaning ones that hit only tumour cells and leave normal tissue alone. Several of these have come into use over the past 15 years and more are in various stages of clinical trials. They’re specific because they knock out the activity of mutant proteins that are made only in tumour cells. Notable examples are Zelboraf® manufactured by Roche (hits the mutated form of a cell messenger – called BRAF – that drives a high proportion of malignant melanomas) and Gleevec® (Novartis AG: blocks a hybrid protein – BCR-ABL – that is usually formed in a type of leukemia).

These ‘targeted therapies’ are designed to not so much to poke the blancmange as to zap it by knocking out the activity of critical mutant proteins that are the product of cancer evolution. And they have produced spectacular remissions. However, in common with all other anti-cancer drugs, they suffer from the shortcoming that, almost inevitably, tumours develop resistance to their effects and the disease re-surfaces. The most remarkable and distressing aspect of drug resistance is that it commonly occurs on a timescale of months.

And being outwitted

Tumour cells use two tactics to neutralize anything thrown at them before it can neutralize them. One is to treat the agent as garbage and activate proteins in the cell membrane that pump it out. That’s pretty smart but what’s really staggering is the flexibility cells show in adapting their signal pathways to counter the effect of a drug blocking a specific target. Just about any feat of molecular gymnastics that you can imagine has been shown to occur, ranging from switching to other pathways in the signalling network to short-circuit the block, to evolving secondary mutations in the target mutant protein so that the drug can no longer stick to it. Launching specific drugs at cells may give them a mighty poke in a particularly tender spot, and indeed many cells may die as a result, but almost inevitably some survive. The blancmange shakes itself, comes up with a counter and gets down to business again. This quite extraordinary resilience of tumour cells derives from the infinite adaptability of the genome and we might do well to reflect that in trying to come up with anti-cancer drugs we are taking on an adversary that has overcome the challenges involved in generating the umpteen million species to have emerged during the earth’s lifetime.

Not the least disheartening aspect of this scenario is that when tumours recur after an initial drug treatment they are often more efficient at propagating themselves, i.e. more aggressive, than their precursors.

Seeing the Invisible: A Cancer Early Warning System?

Sherlock Holmes enthusiasts who also follow this column may, in a contemplative moment, have asked themselves whether their hero would have made a good cancer detective. Answer perhaps ‘yes’ in that he was obsessive about sticking to the facts and not guessing and would probably have said that, when tracking down a secretive quarry, you need to be as open-minded as possible in looking for clues. One of his most celebrated efforts at marrying observation with knowledge was his greeting upon first meeting Dr. Watson: “How are you? You have been in Afghanistan, I perceive”. Watson was suitably astonished by this apparent clairvoyance although its basis was in fact rather mundane and only beyond him because, as Sherlock kindly explained, “You see, but you do not observe.”

Holmes-Image-Loupe

Dr. Holmes perchance?

If Watson had paused to wonder whether Holmes’ combination of superiority complex and investigative genius would have fitted him for a career in the medical fraternity, he might have reflected that indeed many internal afflictions do manifest external signs – much as the furtive body language of a felon on a job might mark him out to the observant eye in the throng of bodies pressing into Baker Street underground station. So perhaps the ’tec turned doc could make it in infectious diseases or become a consultant in rheumatoid arthritis. But would he have steered clear of oncology, reasoning that most cancers are without symptoms during their early development and that even he could not observe the invisible?

Lithograph of Baker Street Station   Baker Street Station on the Metropolitan Railway in 1863 (London Transport Museum collection)

Probably, but before taking that decision he would have asked for a tutorial – perhaps from that bright fellow Stephen Paget, who would have explained that cancers are unusual lumps of cells that can often be cut out by surgeons such as himself. But he’d have highlighted the problem that similar growths commonly turn up later at other, secondary, sites in the body – they are what kills most cancer patients and no one has a clue how this happens or what to do about it. Holmes would doubtless have taken a deep suck on his pipe, commented that, as no one appeared to disagree with William Harvey’s 250 year old finding that blood is passed to every nook and cranny of the body by the circulatory system, it scarcely required his giant intellect to deduce that to be the most probable way of spreading tumours. Further observing that cancers develop very slowly, he would have pointed out that it is highly likely that within the body there might be clues – molecular signs that something is amiss – long before overt disease appears. All that was required was a biological magnifying glass and tweezers to spot and pick out rogue cells and molecules. Muttering ‘Elementary’ he would then have asked to be excused to return to the really tricky problem of outsmarting Professor Moriarty.

An Achilles’ heel?

Well, as we have just reviewed in Scattering the Bad Seed, some 130 years after that imaginary encounter the ‘elementary’ way in which tumours spread to form metastases is just beginning to be revealed and, of course, the hope is that eventually this knowledge will lead to ways of treating disseminated cancers or even preventing them. That’s a wonderful prospect but even more exciting are technical advances enabling us to exploit what Sherlock had spotted as something of a cancer Achilles’ heel – namely that, if tumour cells spread via the bloodstream, we need only the right tools (magnifying glass and tweezers) to detect secondary growths almost before they’ve started to form. As most people know, the earlier cancers are caught the more likely they are to be cured, the most critical intervention being before they have spread to form metastases that are the major cause of death.

The things you find in blood

In fact, quite apart from intact tumour cells migrating around the circulation, it’s been known for 40 years that most types of cell in our bodies have the rather odd quirk of releasing short bits of their DNA into the circulation. Cancer cells do this too and these chromosome fragments reflect the genetic mayhem that is their hallmark. How DNA gets out of the nucleus and then across the outer membrane of the cell isn’t known but it does – and the bits of nucleic acid act as messengers, being taken up by other cells that respond by changing their behaviour. In Beware of Greeks we saw that DNA fragments released by leukemia cells can help those cells escape from the bone marrow into circulating blood.

There’s yet another sort of cellular garbage swishing around in our circulation: small sacs like little cells that contain proteins and RNAs (nucleic acids closely related to DNA). These small, secreted vesicles are called exosomes and in fact they’re not at all rubbish but are also messengers, communicating with other cells by fusing and transferring their contents. So exosomes are another form of environmental educator.

Going fishing

The problem has been that until very recently it has not been possible to fish out tumour cells or DNA from the vast number of cells in blood (we’ve each got over 20 trillion red blood cells in our five litres or so). However, an exciting new development has been the application of silicon chip technology to the detection of circulating tumour cells (CTCs). The chips, which are the size of a microscope slide (10 x 2 cm), have about 80,000 microscopic columns etched on their surface that are coated with an array of antibodies that stick to molecules expressed on the surface of CTCs. By incorporating the chips into small flow cells it’s possible to capture about 100 CTCs from a teaspoon of blood – that’s pulling out one tumour cell from a background of a billion (109) normal cells.

CTC CHIP

Tumour cell isolation from whole blood by a CTC-chip. Whole blood is circulated through a flow cell containing the capture columns (Stott et al., 2010)

This microfluidics approach can also be used to isolate tumour cell DNA. For this the coatings are short stretches of artificial DNA of different sequences: these bind to free DNA in the same way that two strands of DNA stick together to make the double helix.

This remarkable technology may offer both the most promising way to early tumour detection and of determining responses to drugs. It also provides a bridge between proteomic and genomic technologies because DNA, captured directly or extracted from isolated cells, can be used for whole genome sequencing. If this system is able to capture cells from most major types of tumour it will indeed provide a rapid route from early detection through genomic analysis to tailored chemotherapy without the requirement for tumour biopsies. In Signs of Resistance we noted that it’s possible to track the response of secondary tumours (metastases) to drug treatment (chemotherapy) using this method of pulling out tumor DNA from blood and sequencing it.

The really optimistic view is that chip isolation of DNA or tumour cells may be a means to cancer detection years, perhaps decades, before any other test would show its presence. By following up with the power of sequencing, the hope is that appropriate drug cocktails can be devised to, so to speak, nip the tumour in the bud.

Wizard’s secret

By the way, Conan Doyle eventually revealed the method behind Sherlock’s wizardry: Watson was a medical man but walked with a military bearing: the skin on his wrists was fair but his face tanned and haggard and he held his left arm in a stiff and unnatural manner. So here was a British army doctor who had served in the tropics (or somewhere equally hot) and been wounded. In 1886 where would that have been? Oh yes, of course. Afghanistan.

Reference

Stott, S.L., Hsu, C.-H., Tsukrov, D.I., Yu, M., Miyamoto, D.T., Waltman, B.A., Rothenberg, M.S., Shah, A.M., Smas, M.E., Korir, G.K., Floyd, Jr., F.P., Gilman, A.J., Lord, J.B., Winokur, D., Springer, S., Irimia, D., Nagrath, S., Sequist, L.V., Lee, R.J., Isselbacher, K.J., Maheswaran, S., Haber, D.A. and Toner, M. (2010). Isolation of circulating tumour cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences of the United States of America 107, 18392-18397.

POTty training for chromosomes

Our genetic material comes in chunks called chromosomes, the ends of which are capped with repetitive DNA sequences called telomeres. Every time DNA is replicated to make new cells bits of the telomeres are lost – so they get shorter – and eventually this turns on a stress signal that puts a stop to further cell reproduction. So, the older you get the shorter your telomeres become and when that stops you making more cells you conk out. Lurking within our chromosomes is a gene that can stop this happening: it encodes an enzyme called, of course, telomerase that extends chromosome caps. But, you exclaim, a well-known feature of cancer cells is that they are ‘immortal’ – so they must find a way of switching on the telomerase gene that in normal cells is turned off to ensure that we don’t hang around too long. And indeed most of them do – which highlights another of life’s balancing acts: telomerase off = finite life-span, telomerase on = cancer.

Telomeres (red) cap the ends of chromosomes

Telomeres (red) cap the ends of chromosomes

Putting a cap on it

Human telomeres contain thousands of repeats of the 6-base sequence TTAGGG that cap the ends of chromosomes. To prevent these being worn away and enable cells to become ‘immortal’, the genetic mayhem that characterizes tumours usually includes a means of activating telomerase. However, you won’t be surprised to find that extending telomeres is a complicated business and the telomerase enzyme is just one bit of a multi-component molecular machine that does the job. One of the bits is a protein by the name of POT (POT1 to be precise) and a Spanish group have just shown that mutations in POT1 occur in chronic lymphocytic leukemia. Normal POT1 acts as a negative regulator that suppresses telomere extension: mutations in POT1 permit telomere extension and also enable chromosomes to fuse end-to-end with one another – a common type of genetic damage in leukemia. It appears that, although POT1 mutations are quite rare, they occur only in the clinically aggressive subtype of CLL – so they provide not only a new potential drug target but also a prognostic indicator.

Incidentally, despite what you might think, ‘cancer genes’, i.e. genes that by acting abnormally (as a result of suffering some sort of mutation, either in themselves or indirectly) can help to drive cancer development, have names that are very sensible and logical. Thus POT1 stands for protection of telomere – and it’s POT1 just in case a close rello turns up – which would be POT2.

Reference

Ramsay, A.J. et al., (2013). POT1 mutations cause telomere dysfunction in chronic lymphocytic leukemia. Nature Genetics 45, 526–530.

http://www.readcube.com/articles/10.1038/ng.2584?locale=en

Unkinking Kindle

In response to a wonderfully appreciative email about the book I’m posting the pictures (some in colour!!) because the reader couldn’t get Kindle to show them – although my publisher’s digital book manager cannot find any problem with the files.

Photographs (Plates 1 to 10) in Betrayed by Nature:

Plates 1 and 2

Plates 3 and 4

Plate 5

Plate 6

Plate 7

Plate 8

Plate 9

Plate 10

Genetic Roulette in a New World

In 2003 it was a sensation. No really – it’s probably true that in medicine only the first human heart transplant operation back in 1967 has generated as much publicity. That was in the pre-web dark age but, nevertheless, the South African surgeon Christiaan Barnard was immortalized as a global hero: even the patient’s name was on everyone’s lips (Louis Washkansky if you’re struggling to recall) and you can re-live the whole event at the Groote Schuur Hospital museum in Capetown. But, although 2003 was just a decade ago, in today’s world sensations fade almost with the following dawn, whether they are pop groups or life-changing scientific advances.

So if now you mention “The Human Genome Project” to a man on the Clapham omnibus you are likely to elicit only a puzzled look. What happened in 2003 was of course that the genetic code – that is the sequence of bases in DNA – was revealed for the entire human genome. And an astonishing triumph it was, not least because, in contrast to almost everything else in history with a major British component, it was completed within schedule and under cost.

The feat was deservedly greeted with a fanfare of public interest unprecedented for any scientific project short of the early space missions. President Clinton in the White House was hooked-up live to whoever was living in No. 10 at the time, the leading British scientists in this amazing project dropped in for tea and Mike Dexter, then Chairman of The Wellcome Trust and a restrained and conservative fellow – being a scientist – described it somewhat inelegantly as “… the outstanding achievement not only of our lifetime, but in terms of human history.”

The Sanger Centre, Cambridge

The Genome Analysis Centre, Norwich

The Genome Institute at Washington University

However, even more remarkable is what happened next. The ensuing decade has brought technical advances so breathtaking as to almost overshadow the original human genome project itself. This quite staggering revolution has seen the introduction of fully automated, high throughput flow cells that simultaneously carry out hundreds of millions of separate sequencing reactions – just say that slowly. In the jargon it’s called ‘massively parallel sequencing’. The upshot of this stunning technology is that sequencing speed has gone up by 100 million times whilst, almost unbelievably, the cost has dropped by a factor of 10,000. Even computing science can’t match that progress!

One consequence of this incredible, though relatively unpublicised, revolution is that genomes can be now be sequenced on an industrial scale and in the years to come that is going to impact on every facet of mankind’s existence. Thus far the field of cancer has been the foremost recipient of this technological broadside with thousands of tumour genomes now sequenced. This has unveiled the almost incomprehensible panoply of genetic changes that cells can sustain and yet emerge still capable of proliferating. One of the first cancer genomes to be sequenced was that of a female who had died from leukemia. The work was carried out by The Genome Institute at Washington University in St. Louis, Missouri and since then, under its Director Richard Wilson, this group has continued to be a world leader in genomics and in particular in unravelling the extraordinary complexity of the group of cancers collectively called leukemias.

Wilson and his colleagues know, of course, that they are at the forefront of the most extraordinary transformation in medicine – because eventually it will affect everyone –though Rick Wilson himself is as improbable a revolutionary as you could imagine: a gentle, soft-spoken American, he’s what on this side of the pond would be called a thoroughly nice chap.

However, if they had any doubts about the direction in which their science was leading the world, these would have been dispelled when one of their own community, Lukas Wartman, was diagnosed with a very rare form of leukemia. This had first appeared ten years ago when Lukas was a student completing his medical degree at Washington University, and at that time it had been treated with chemotherapy and a bone-marrow transplant.

In the following years, Dr. Wartman had pursued his career goal of becoming a practicing oncologist specializing in leukemia until, in July 2011 the disease returned and he went into relapse. As his condition deteriorated rapidly and only one outcome seemed possible, those treating him turned in desperation from conventional approaches to local expertise. They applied genomic analysis to his cancer cells. From the vast number of disruptions identified, one in particular stood out: an abnormally expressed gene that had previously been associated with other types of leukemia but is very rare in the form Wartman had developed.

By an unlikely chance there is a drug available that can knock out the activity of the protein made by that gene. Its effect was phenomenal, restoring the normal blood count and achieving complete remission. This wonderful outcome does not mean that Dr. Wartman is cured for life – but for now he is alive and well – and a co-author of the group’s latest paper – on leukemia.

He had been a desperately unlucky in that the genetic roulette that is life generated in him a hand of mutations that drove the development of a rare and almost invariably lethal form of leukemia. But life also smiled on Lukas Wartman in that circumstances found him at the heart of the genomics revolution that is ushering in a new world of medicine. His isn’t the first life to be saved through the use of this fabulous technology but he is one of the first few who will, in years to come, be followed by many as these marvellous methods for diagnosis and the design of treatment come into widespread use.

Cooperative Cancer Groupies

Few words carry more impact than the gentle syllables of cancer. Transform it into any language, its effect is unchanged: cancer, cancro, Krebs, рак … Always that inner tightening as we prepare ourselves for something we’d prefer not to hear. And yet, and yet … like so much of life there is an obverse – more than one in fact. Down the years few things have revealed more of the greatness of the human spirit – the fortitude, resilience and compassion of which mankind is capable. And it’s also a wonderful thing because, whatever its downsides, without cancer we would know far less about the amazing flexibility and adaptability of nature. Endless beautiful examples have been teased from its mysteries by inquisitive, curious and sometimes plain lucky scientific detectives.

Of late a particularly fertile facet has been what one might call the supporting cast: not tumour cells themselves but families that have moved in next door. If the idea that both normal and abnormal may play a role seems a bit strange, recall that, as well as being wonderful, cancer’s also funny – peculiar, that is – in being generated within ourselves: something goes wrong with cells that are perfectly normal and the result is something unusual. A new growth. A neoplasm. And our body reacts as it almost always does when something odd happens: it sends reporters along to find out what’s happening and put a stop to it. These roving sleuths are cells of the immune system – collectively white cells.

As with almost any unusual event – a new kid in school, a spotted celeb, a traffic accident – a crowd has started to gather round. In the body it’s called inflammation and it’s the first sign that our immune response is being switched on. The cellular groupies that turn up at the earliest signs of a tumour are a motley lot: all the broadsheets and tabloids are there, so to speak. But they differ from human onlookers in that each has a job to do. The first response is that some of the groupies release chemical signals that can target tumour cells for destruction by other groupies. The tumour is seen as ‘foreign’, just like an infection, and the response is ‘get rid of it’. We have no idea how efficient this kind of tumour elimination is but we might guess it’s not bad as most cancers don’t appear until we’ve been around for over 60 years.

When a tumour does manage to grow to a detectable size, that protection has clearly been overcome. But astonishingly, when this happens it’s not merely that the anti-tumour armoury has failed. It’s worse than not having enough fire-power: it’s actually been subverted, perverted if you wish, ‘turned’ as John le Carré might say, so that the immune cells that set out as assassins have become genuine groupies. Now the chemical signals they throw onto the tumour stage support growth and protect the cancer cells from destruction. Normal cells, recruited to the scene of cellular abnormality, have become in effect part of the tumour, essential for its survival and continued growth.

A stunning example of the tumour cooperative happens in chronic lymphocytic leukaemia. The leukaemia cells are a typical tumour in that their metabolism is abnormal. One upshot of this is that they make a lot of very reactive things called free radicals that are toxic – that is, will kill the leukemia cells unless they can make a neutralizing chemical called A. But to make A they need a building block B. B needs a carrier to get across the outer membrane into cells and the leukemia cells don’t make that carrier. But one of the ‘groupies’ does: it takes up lots of B, turns it into C and then pumps that out so that the tumour cells are bathed in C – which they can take up. The leukemia cells convert C into B, then make A, which knocks out their free radicals – so they survive and the tumour grows. If you can describe a thriving tumour as wonderful, it’s jaw-droppingly clever. And it’s not all bad news because blocking the transfer of C offers a new drug target for treating the most common adult leukemia in the Western world.

Reference

Zhang, W. et al., (2012). Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nature Cell Biology 14, 276-286.

Obesity and Cancer

Science, you could say, comes in two sorts. There’s the stuff we more or less understand – and there’s the rest. We’re pretty secure with the earth being round and orbiting the sun, the heart being a pump connected to a network of tubes that keeps us alive, DNA carrying the genetic code – and a few other things. But human beings are curious souls and we tend to be fascinated by what we don’t know and can’t see – why the Dance of the Seven Veils caught on, I guess.

Scientists are, of course, the extreme example – they spend their lives pursuing the unknown (and, as Fred Hoyle gloomily remarked, they’re always wrong and yet they always go on). But in this media era they pay a public price for their doggedness because they get asked the pressing questions of the moment. Is global warning going to finish us off soon, why is British sport generally so poor and – today’s teaser – does being fat make you more likely to get cancer?

A few facts go a long way

The major cancers have become familiar because the numbers afflicted are so staggering – but the one good thing is that the epidemiology can tell us something about the disease. Thus for cancers of the bowel, endometrium, kidney, oesophagus and pancreas and also for postmenopausal breast cancer there is clear evidence that being overweight or obese makes you more susceptible. In other words, if you compare large groups with those cancers to equally large numbers without, the disease groups contain significantly more people who are fat. We should add that the above list is conservative. A number of other cancers are almost certainly more common in those who are overweight (brain, thyroid, liver, ovary, prostate and stomach tumours as well as multiple myeloma, leukaemia, non-Hodgkin lymphoma and malignant melanoma in men).

Sizing up the problem

The usual measure is Body Mass Index (BMI) – your weight (in kilograms) divided by the square of your height (in metres). A BMI of 25 to 29.9 and you’re overweight; over 30 is obese. In England in 2009 just over 61% of adults and 28% of children (aged 2-10) were overweight or obese and of these, 23% of adults and 14% of children were obese. And every year these figures get bigger.

How big is the risk?

Impossible to say exactly – for one thing we don’t know how long you need to be exposed to the risk (i.e. being overweight) for cancer to develop but in 2010 just over 5% of the total of new cancer cases in the UK was due to excess weight. That’s another conservative estimate, but it means at least 17,000 out of 309,000 cases, with bowel and breast cancers being the major sites.

What’s going on?

Showing an association is a good start but the important thing is to find out which molecules make that link. For obesity and cancer detail remains obscure but broad outlines are emerging, summarised in the sketch. In obesity fat (adipose) cells increase in both number and size (so it’s a double problem: more cells – and the fat cells themselves are fatter). As this happens other cells are recruited to adipose tissue and, from this cellular cooperative, signalling proteins are released that have the potential to drive tumours. This picture is similar to that of the microenvironment of tumours themselves, where many types of cell infiltrate the new growth. Initially this inflammatory and immune response aims to kill the tumour but if it fails the balance of signalling shifts so that it actually helps the tumour grow. In addition to signals from fat cells themselves, obesity is usually associated with increased levels of circulating growth hormones (e.g., insulin) and of lipids, both of which may also promote tumour development.

Thus many signals with cancerous potential arise in obese individuals. In principle these could initiate tumour growth or they could accelerate it in cancers that have started to develop independently of obesity. So it is complicated – but at least as new signalling strands emerge they offer new targets for drug therapy.

In obesity abnormal signals from fatty tissue can combine with others arising from perturbed metabolism to help cancers develop

Reference

World Cancer Research Fund (WCRF) Panel on Food, Nutrition, Physical Activity, and the Prevention of Cancer (WCRF, 2007).