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On Randomness, Determinism, False Dichotomies and Cancer

Before I start – a short summary

[1] A recent paper attributed a large proportion of variation in incidence of cancers across different tissues to the number of stem cell divisions in them, and
stochastic errors in cell division.

[2] The paper grouped tumour types with known external causes as “deterministic” and those without as “stochastic”

[3] I have seen people being hostile to the notion of stochasticity in cancer who’ve postulated other deterministic factors, with the implicit assumption that what is stochastic is really deterministic processes with as-of-now undiscovered causes.

[4] Here I explain why processes with known causes are still stochastic, leading to my gripe with both the misunderstanding that has permeated discussion of the paper as well as the iffy notion of grouping tumours into stochastic and deterministic ones in the paper. My assertion is that even those cancers strongly driven by external carcinogens involve randomness/stochasticity.


Sooo, last week, a paper was published in the journal Science that linked the number of stem cell divisions in normal tissues to the rates of incidence of cancer in that tissue. So the more dividing stem cells there were in a tissue, it turns out, the more likely the tissue would be prone to developing cancers in populations.

The paper is to be found here and where I quote without further reference, it is from this paper

To quote, the abstract reads…


Some tissue types give rise to human cancers millions of times more often than other tissue types. Although this has been recognized for more than a century, it has never been explained. Here, we show that the lifetime risk of cancers of many different types is strongly correlated (0.81) with the total number of divisions of the normal self-renewing cells maintaining that tissue’s homeostasis. These results suggest that only a third of the variation in cancer risk among tissues is attributable to environmental factors or inherited predispositions. The majority is due to “bad luck,” that is, random mutations arising during DNA replication in normal, noncancerous stem cells. This is important not only for understanding the disease but also for designing strategies to limit the mortality it causes.

 Much of the reaction I’ve seen to the paper on the cybersphere involves a fundamental misunderstanding of the processes that drive cancer – far too many people have been thinking that things cause cancers deterministically ,and even in the paper the authors group cancers into stochastic ones and deterministic ones in Figure 2, somehow conveying the impression that there are those that are caused, and those that are due to chance. There are several well-described summaries for laypeople already on the web, ranging from the almost always excellent David Gorski’s post , to PZ Myers’ explanation of the paper . David’s post in particular describes the trainwreck that the media misinterpretation of stochastic errors as “bad luck” has led to.

To summarise all of that – the paper says that differences in the incidence of cancers amongst different tissues can be mostly explained by the number of cell divisions in stem cells, and known environmental factors and genetic predisposition only explain a very small percentage of why different tissues get cancers at different rates. They postulate that mutations accumulate with the number of stem cell divisions because of stochastic or chance errors in cell division.

This led David Colquhoun, on twitter, to note that a lot of the opposition to this finding seemed to be from people who opposed the role of chance in driving cancers…and he is right about the amazing indignance of those reacting with hostility to the role of chance , for reasons I will tell you in a little bit.


Additionally, there were people positing the notion that it couldn’t be chance, there was just some undiscovered latent factor/factors – and so the dichotomy was set up between stochastic (assumed to be with no cause) and deterministic (assumed to be with causes) in what passed for discourse amongst those that did protest too much.

Where the paper gets it right…

Coming to the paper itself, there are bits I like – it was quite elegant evidence for the role of stem cell divisions in driving the evolution of cancer; in some cases tumours can be latent for a long long time before they present clinically; and previous studies have reported a case of latency in lung cancer for up to two decades before the tumour showed up. Turns out you need multiple mutations to go from a normal cell to a cancerous cell , and obviously cellular lineages that persist longer (and have more divisions) are likelier to acquire the full complement of mutations.

What I disagree with is the paper is the authors lumping everything that is not attributable to external factors to be the product of stochastic errors in cell division.

Importantly, my gripe with that is not that they are attributing it to a stochastic process, because, as I shall explain, almost all mutations, even those with known causes are still random. My problem was with them putting it down to cell division and DNA replication ; turns out there are loads of internal cellular processes, which are neither genetic nor products of environmental factors, that can generate mutations – of course, all of these are still stochastic, but their phrasing of everything as errors in cell division is something I find too vague.

Some of these internal processes include age-related mutations, which are characterised by the spontaneous deamination of CpG dinucleotides, and by and large comprise a mutational signature that is found ubiquitously across cancers of different types.

Wondering what a mutational signature is? Well, DNA is made of 4 bases, and when you look at what mutations (DNA sequence changes) have taken place in tumours compared to normal tissue you can look at the DNA sequence around the mutated sequence, and turns out certain processes generate mutations in certain sequence contexts; I’ve blogged about this earlier in the context of APOBEC enzymes, which accidentally mutate human DNA and can potentially cause cancer.

So, the point I am trying to make here is that every mutation that isn’t caused by inheritance or exposure to external carcinogens are not down to errors in DNA replication during  cell division , unless you use the term for all mutations generated internally, in which case it loses nuance. Indeed, there is extensive documentation of internal mutagenic processes, the repair pathways that deal with the lesions they produce, and so on and so forth…

However, this is the important bit – Even if causes are known, external or internal, this does not mean they are deterministic; I reiterate, mutations with known causes are still random and chance still plays a massive role.

How can things have causes and yet be random?!

The trouble is that the popular use of the word “random” differs from the scientifically and statistically rigorous usage of the term thereof. In common parlance, people often assume that random means “for no reason” or “with no cause” , like “She turned up wearing a hat, totally random, blud”.

In science, it means “following a probability distribution”, where uncertainty is involved. This is why we talk of risks – see, smoking causes lung cancer and there is a mutational signature associated with smoking, and many of the mechanisms of mutations induced by cigarette smoke are well known. However, the relationship between smoking and lung cancer is not deterministic – i.e, not everyone who smokes heavily gets lung cancer – what smoking does is it increases the chances one has of getting lung cancer. This is why the relationship between lung cancer and smoking is stochastic (chance is involved).

Likewise, APOBEC enzymes can cause very specific mutations and have a specific mutational signature associated with them, i.e, they change C to T or C to G when there is a T before and a G,A or T after; i.e, TCW -> TTW or TGW – however, the mutations induced by APOBEC enzymes are still random.

How can this be the case?

Well, it turns out, that for cancers to develop, you need mutations or epigenetic changes in certain types of genes – those that control the cell’s (or in this case, a lineage of cells’) ability to acquire the hallmarks of cancer (i.e, ability to grow without external stimulation, ability to escape cell death, ability to escape the immune system et cetera)  and not all regions of the human genome harbour genes that can cause cancer. So there are, for instance , plenty of TCW sites in the genome that are not capable of affecting cancer-associated genes if they pick up an APOBEC induced mutation.

So while we know that a molecule of APOBEC can act upon a TCW site to mutate it at a given rate – which TCW site in the genome gets mutated is down to chance, and the probability it gets repaired also involves a chance element; this is how even a factor with a well-defined mode of action can still make random mutations.

On top of this – you see chance involved when different combinations of mutations occur in a cell or its lineage – the right combination of cancer-causing mutations happening is still a matter of chance – whether it evolves sufficiently to evade the immune system is still a matter of chance; chance is everywhere – cancer evolution is a stochastic phenomenon, fundamentally.

Additionally, mutations happen at different sites in the genome with different probabilities, but if it happens in a cancer related gene that then gives cells that carry it a selective advantage or not is a matter of chance – this is why cancers contain both driver mutations that confer growth advantages and passenger mutations which don’t.

This leads me to my main bone of contention with the paper, along with the iffy statistics of the second figure in the paper, I find the authors group them into stochastic and deterministic classes ;

They also make this clanger

We refer to the tumors with relatively high ERS as D-tumors (D for deterministic; blue cluster in Fig. 2) because deterministic factors such as environmental mutagens or hereditary predispositions strongly affect their risk. We refer to tumors with relatively low ERS as R-tumors (R for replicative; green cluster in Fig. 2) because stochastic factors, presumably related to errors during DNA replication, most strongly appear to affect their risk.

It turns out that they are all stochastic – because, for instance where exactly in the genome smoke-induced carcinogens induce mutations is down to chance. Smoke and environmental mutagens are not deterministic factors, nor are any internal mutational processes. 

How HPV driven cancers get their mutations…

Hi there!

It’s been a long time since I last blogged, but that is because I’ve been swimming round in data, which has incidentally led to the findings that were published in this paper , which I will describe in this post.

HPV and the link to cancer.

HPV (Human Papillomaviruses) consist of a family of viruses that infect keratinocytes (skin cells) that line the outside of the body and the inner cavities – some of them just cause warts (and genital warts) but some of them are capable of driving the formation of cancer. These types, which are called “High-risk” strains, are the ones that are targeted for prevention by HPV vaccines.

High-risk HPV strains differ from low-risk strains in terms of cancer-causing ability because of proteins they make during their life cycle. Cells need to be actively dividing to permit HPV replication and in order to do this, the virus uses two proteins, called E6 and E7 , to block and degrade two proteins in human cells, called TP53 and pRb, which are two potent tumour suppressors (genes that prevent tumour formation).

Normally, E6 and E7 are only active for a brief while during the virus’ life cycle, which culminates in the production of more viruses that restart the cycle all over again, but before HPV driven cancers form something very strange happens; by complete accident the viral genome gets inserted and integrated into human DNA in infected cells, or infected cells get locked into a state where E6 and E7 are produced all the time. Suddenly you’ve got cells with TP53 and pRb off all the time, leaving behind cells that can grow abnormally. We see this when women have cervical scrapings looked at, and see “dysplastic” cells that have grown clumpy and abnormal.

However, these dysplastic cells are not cancerous – and haven’t acquired all the hallmarks of cancer. For this to happen there need to be additional changes to the DNA sequence (Mutations) of the genes in dysplastic cells that can confer those properties. Well known examples of things that cause mutations include tobacco smoke; for quite a while it had been an open question as to where HPV-driven tumours got their mutations from.

Suspicions are aroused: could the APOBEC family of proteins be making these mutations? 

One of my major research interests is to see what genes are expressed more and what genes are turned off in HPV driven cancers, and when defining a signature for these tumours I compared them to normal tissue and HPV negative tumours that arise in the same tissue (while cervical cancers usually all tend to be HPV-driven, there are head and neck cancers caused by HPV and those caused by chronic tobacco and alcohol exposure) and one of the genes that I found expressed at high levels in HPV-positive tumours was APOBEC3B.

APOBEC3B is one of many proteins of the APOBEC cytosine deaminases family. These act either on RNA or DNA when it is a single stranded state, and take part in the body’s immune response against viruses by messing up the RNA/DNA from the viruses. They work by changing cytosines, one of the four bases that make up DNA to uracil (a base that is normally only found in RNA) which then gets converted to a thymine or a guanine (two other bases that make up DNA); so if you get lots of these changes in viral DNA you fundamentally break them so they can’t do any of the things they usually do, and it had been known for a while that you could find HPV with messed up DNA in precancerous lesions with patterns of change associated with APOBEC proteins.

This led us to wonder if APOBEC proteins could end up accidentally changing human DNA just like it would change viral DNA and therefore generate the necessary DNA sequence changes to cause cancer; and at the same time we started wondering that a couple of papers came out showing that there were human cancers in which mutations looked like they were being generated by APOBEC enzymes, very likely APOBEC3B (We could tell it was likely APOBEC 3B because it is known to change cytosines that are preceded by a thymine and followed by guanine or adenine or thymine, so if the sequence was TCA or TCG or TCT it would be converted to TGA/TTA or TGG/TTG or TTT/TGT ). There is an alternative process that can also generate TCG->TGG/TTG mutations, so in order to specifically measure APOBEC activity we ended up using the others, which we referred to in the paper as TCW to TKW (TCW->TKW, where K = G or T and W = A or T).

Those previous papers also noted that cervical cancers had lots of mutations that showed the APOBEC signature, but the question remained – was this down to it being the cervix? or was it down to these tumours being HPV+? We decided to take a look in head and neck cancers as well where we could compare HPV+ and HPV- tumours that arose in similar tissues to see if there was truly an association with HPV, and hence we did the work reported in the paper…

HPV positive tumours have a vastly higher fraction of mutations belonging to the APOBEC signature.

First, we ended up looking at levels of APOBEC mutagenesis and how much of all the mutations in tumours were attributable to them using publicly available data for 40 HPV+ head and neck tumours and 253 HPV- head and neck tumours. To do this we used multiple approaches – including looking at TCW->TKW mutations and also trying to break down all the mutations we see in these tumours into patterns of mutations, as was done by these people at the Sanger Institute , and also looking at enrichment for the TCW->TKW mutation pattern locally. All the approaches we used showed the same thing – HPV+ tumours had a vastly higher proportion of mutations most likely caused by APOBEC enzymes.

Figure1:APOBEC mutations are highly enriched in HPV+ HNSCs

Multiple measures of APOBEC activity showed a strong association with HPV status but not age or smoking; APOBEC, age and smoking were the three processes we identified as driving the signatures using the Sanger Institute’s approach. The more the numbers are shifted to the right the stronger the association with the factor listed on the left. 

We found signatures previously associated with APOBEC, smoking and age, and showed that APOBEC activity was not associated with the latter two, which was as expected. Having identified an association with HPV driven tumours we wanted to know if this was a general antiviral response or something HPV specific…so we took a look at patterns of mutations in liver cancers caused by hepatitis B and C viruses and found no evidence for APOBEC mediated mutations being significantly enriched in these tumours.

Of drivers and passengers

Most tumours have hundreds and thousands of mutation, but only a few actively contribute to the acquisition and maintenance of the hallmarks of cancer. So, having initially identified high proportions of APOBEC-mediated mutations in HPV driven cancers when looking across the exome (all protein coding genes in general) we decided to ask if the enrichment we saw in all genes was also maintained when we restricted our searching to genes known previously to drive cancer or those that share features associated with drivers, like occurring at a frequency greater than expected by chance. Our analyses confirmed that APOBEC-mediated mutations were again enriched in the HPV+ head and neck, and cervical cancers compared to the HPV- HNSCs.


Differences between HPV negative HNSCC and HPV+ tumours (HNSCC and Cervical cancer) are maintained when looking at all protein-coding genes (whole exome) and likely driver mutations (MutSig).

Then we went on to look at which driver genes happened to be most mutated by APOBEC proteins, and found a gene called PIK3CA (one of the components of a protein complex called PI3 kinase) towards the very top of the list. PIK3CA has previously been reported as being vital to the sustenance of many HPV positive tumours in particular and head and neck cancers in general, and drugs are being developed to target it. Interestingly, we observed that in the HPV+ tumours 22/25 PIK3CA mutations recorded were of the APOBEC type, while this wasn’t the case for the HPV negative tumours.

This then led to yet another question – can the levels of APOBEC activity explain a preference for APOBEC mutations in HPV-positive tumours? Now for driver genes there are two things that may govern what kinds of mutations we see – how much of a growth advantage a mutation in a driver gene gives that cell and the mutation itself. My supervisor, Tim Fenton, who worked on PI3 kinases previously, knew that there were two regions in PI3 kinase amongst which mutations regularly occurred (one or the other) and then realised that one of them contained a TCW sequence that APOBEC proteins could act on while the other one did not.

The PIK3CA gene makes a protein called p110-alpha, and proteins have different distinct elements in their structure, called domains. One region, called the helical domain, is often mutated at two TCW sequences while the other region, called a kinase domain, is not, and both mutations confer similar growth advantage, and if you look across multiple tumour types, overall you tend to see a 50-50 split between the two. This enabled us to account for growth advantage and directly see if APOBEC activity, which we had already measured by looking at all protein-coding genes, and a preference for APOBEC-induced mutations in the helical domain, were linked.

Since PIK3CA is mutated in multiple types of cancers, I was able to grab some data from The Cancer Genome Atlas project and measure how strongly there was a skew towards acquiring helical domain mutations compared to the kinase domain mutations and just look at what APOBEC activity looked like in each of those types of tumours. The results were quite robust – the higher the APOBEC activity in a cancer type, the stronger the preference for helical domain mutations compared to kinase domain mutations.


Figure 3. A – as you move from left to right (tumour types are arranged from left to right based on median APOBEC activity), you see helical domain mutations (black bars) become strongly preferred compared to kinase domain mutations (yellow bars). B – plotting the median TCW->TKW fraction (APOBEC activity) against the proportion of PIK3CA mutations that are helical hotspot mutations shows a strong correlation.

So yeah, people had been wondering why in bladder cancers, for example, you saw such a strong preference for helical hotspot mutations – we basically addressed that long-standing question with these analyses.

Explanatory factors

So the one other thing we did was to look at what might be driving this process, and surprisingly we found no correlation between how much E6 and E7 was being expressed in these tumours and APOBEC activity, or for that matter between APOBEC3B gene expression and APOBEC activity, and did find a strong link with how many mutations in total these tumours had. The work has led us to hypothesize it may be something like DNA damage induced by HPV, that generates the substrate for APOBEC3B to act upon, that drives the process.


Our work suggests that HPV positive tumours evolve in a trajectory where they incorporate HPV DNA into their own, leading to sustained E6/E7 expression, followed by APOBEC activity until a driver mutation occurs, after which clones expand and show the APOBEC signature when their DNA is sequenced while in HPV negative HNSCC smoking and alcohol do this job, and if PIK3CA is the gene mutated the HPV positive tumours tend to have helical domain hotspot mutations because APOBEC proteins are responsible for them…

Additional stuff

The journal did a Q&A that expands on some of the work in the paper, and you may find it here .

There is a press release from UCL here.


India’s Supreme Court has a homophobic brain fade…

It looks like people in positions of power in the Indian judiciary are not particularly exempt from indulging in confused thinking to any degree. Just two years ago, the Delhi High court decided to decriminalise homosexuality in what was a very progressive and welcome move, ruling its criminalisation on the basis of a law from the 1860s unconstitutional on grounds of being discriminatory.

Today, India’s Supreme Court decided to overturn that decision and recriminalise homosexuality in the country, upholding it to be constitutional and saying that Parliament would have to amend the law if they had to get rid of it, which the government is not saying it will do with elections coming up next year and voting along religious lines being an important part of the equation; what is beyond my comprehension though is how they could have possibly found it constitutional given this fundamental right in the Indian constitution; that every citizen shall have

  1. Right to equality, including equality before law, prohibition of discrimination on grounds of religion, race, caste, sex or place of birth, and equality of opportunity in matters of employment, abolition of untouchability and abolition of titles.”

In what parallel universe would the assertion that you can only have consensual sexual relationships with a woman only if you are a man and consensual sexual relationships with a man only if you are a woman not be discriminatory on the basis of sex?

To quote their judgement as reported in [1]

“It is relevant to mention here that Section 377 IPC does not criminalize a particular people or identity or orientation. It merely identifies certain acts, which if committed, would constitute an offence. Such prohibition regulates sexual conduct regardless of gender identity and orientation,” Justices Singhvi (sic) said. “

Yeah, how about we take it a bit further and say “This law does not criminalise a particular group, tribe or gender, it only criminalises their act of breathing!”; reductio ad absurdum it may be but it highlights the vapidity of the whole rotten affair. What particular acts are they talking about, anyway?

Anal sex? Are they going to check if straight couples too aren’t doing it? Fingering, fellatio, tribadism?  I despair at the lack of logic involved here, it is also quite trivial to imagine any number of acts that are integral to the well being and equality of various people and groups of people – one can’t then ban that act and then say it isn’t discriminatory because it is that act which is banned; what matters is that it can have disproportionate impact on a subsection of the population and it can become discriminatory towards them as a direct consequence… 

More nonsense from them.

“”While reading down Section 377, the division bench of the HC overlooked that a miniscule fraction of the country’s population constitute lesbians, gays, bisexuals or trans-genders and in the last more than 150 years less that 200 persons have been prosecuted for committing offence under Section 377 IPC and this cannot be made a sound basis for declaring the section ultra vires (violative of) the provisions of Articles 14, 15 and 21 of the Constitution,” the apex court said. ”

So unconstitutional laws are constitutional when the people they affect [1] comprise a small minority of the population and [2] it doesn’t get used to persecute them  very frequently, which is completely against the fact that fundamental rights apply to every citizen; not just those belonging to groups that are of more than a miniscule size or are persecuted at more than some arbitrarily determined frequency.Seriously?

The people who’ve welcomed its recriminalisation have mostly done so on the basis of religious beliefs and an appeal to tradition; I wonder if they will also welcome a return to widow-burning and child marriage in the name of the latter – it comes across to me as an unabashed display of egregious stupidity and contemptuous inhumanity, as does the ridiculous yet alarming bilge on display in the comments section in the article I quoted excerpts of the judgement from.

As the holder of an Indian passport I hate being associated with regressive fuckwits, and the guardian of India’s constitution has shown itself capable of mental gymnastics that would make an invertebrate proud. For shame…



PS- This article on the Daily Beast rips the judgement apart better than I could and highlights more flaws and fallacies. Go read it. 

Universities UK endorses gender segregation in a blatant display of affection for religious privilege.

Right, so UCL ended up banning an organisation of Islamists from holding debates after they attempted to enforce gender segregation at an event they hosted using the university’s facilities . Since then, Universities UK has come up with a judgement about segregation policies at universities, and shock horror they’ve shown all the cognitive abilities of anencephalic sea sponges.

I quote the petition page on Avaaz for some of the more alarming bits of the nonsense that has come to be espoused by them.

“Universities UK (UUK) has issued guidance on external speakers saying that the segregation of the sexes at universities is not discriminatory as long as “both men and women are being treated equally, as they are both being segregated in the same way.”

UUK add that universities should bear in mind that “concerns to accommodate the wishes or beliefs of those opposed to segregation should not result in a religious group being prevented from having a debate in accordance with its belief system” and that if “imposing an unsegregated seating area in addition to the segregated areas contravenes the genuinely-held religious beliefs of the group hosting the event, or those of the speaker, the institution should be mindful to ensure that the freedom of speech of the religious group or speaker is not curtailed unlawfully.”

I wonder if they’ll say the same about discrimination on racial lines; the fact there is no evidence to support that proposition but there is evidence to support the notion that they will endorse segregation by gender has well and truly been provided; that tells us everything we need to know – that it is ok to treat people differently by the fruits of their karyotypes but discrimination by skin colour is taboo. The case for me illustrates two major biases; sexism at first and secondly, religious privilege.

The rampant sexism involved in segregation by gender here is trivially simple to observe, the other point though has major problems associated with it and the implementation of the whole policy dreamt up in careless fashion by the powers-that-be at Universities UK is going to be quite tricky, even if in some parallel universe by dint of dictionary gymnastics it weren’t sexist;

I shall quote the relevant bits again;

“and that if “imposing an unsegregated seating area in addition to the segregated areas contravenes the genuinely-held religious beliefs of the group hosting the event, or those of the speaker, the institution should be mindful to ensure that the freedom of speech of the religious group or speaker is not curtailed unlawfully.”

So just believing in a deity gives you the right to trample over the rights of those who don’t claim divine sanction for beliefs that are equally well grounded at the very least, and way better at best; I wonder what will happen if some of us turn up having had a spontaneous revelation of our own from the god of egalitarianism and being segregated would be an affront to our genuinely held “religious” beliefs. Do these people have any ability to think straight at all? What if someone had similarly strongly held religious beliefs calling for racial segregation, or homophobic segregation?

The insinuation is that somehow religious beliefs are to be privileged over non-religious ones. Of course, looking further at the quote; not only do they suggest that segregation is permissible, but also suggest that the provision of an unsegregated area is *also* at the discretion of the hosts, which would, even if there were some kind of religious right to segregation to be granted legitimately, would still trample over the rights of those that don’t want segregation just because the former is religiously motivated.

There is a petition going round on Avaaz calling for Universities UK to rescind their ill-conceived erroneous vomitings laden with the toxic fruits of religious bigotry and privilege, and if you care about pushing back I think you should sign it too,  here;


Update: Universities UK has come up a statement in its defence, saying they do not condone gender segregation, and the guidelines were part of a case study recommending how universities may, subject to their own autonomy, accommodate the wishes of sexist bigots should they end up speaking at universities while not getting into trouble with the law; the implications of this statement extend further than to Universities UK as a consequence – that the law privileges bigotry to the point that some of the feasible solutions proposed by UUK (i.e, the stuff mentioned above) are legally permissible or necessitated by the way British laws are. We’ve gone from a “UUK messes up” situation to a “legal loophole needs dealing with” situation; not sure if that makes things even worse.

The statement is to be found  here

HPV vaccines, cervical cancer and conspiracy theories…

Right, thanks to a friend I had my attention drawn to this article

I fully intend to take apart the dubious claims made by the article and illuminate where those claims are grounded in plain ignorance. Regarding the report the report quotes, which follows below…

“Dr. Harper explained in her presentation that the cervical cancer risk in the U.S. is already extremely low, and that vaccinations are unlikely to have any effect upon the rate of cervical cancer in the United States.  In fact, 70% of all HPV infections resolve themselves without treatment in a year, and the number rises to well over 90% in two years.  Harper also mentioned the safety angle.  All trials of the vaccines were done on children aged 15 and above, despite them currently being marketed for 9-year-olds.  So far, 15,037 girls have reported adverse side effects from Gardasil™ alone to the Vaccine Adverse Event Reporting System (VAERS), and this number only reflects parents who underwent the hurdles required for reporting adverse reactions.  At the time of writing, 44 girls are officially known to have died from these vaccines.  The reported side effects include Guillian Barré Syndrome (paralysis lasting for years, or permanently — sometimes eventually causing suffocation), lupus, seizures, blood clots, and brain inflammation.  Parents are usually not made aware of these risks.  Dr. Harper, the vaccine developer, claimed that she was speaking out, so that she might finally be able to sleep at night.  ’About eight in every ten women who have been sexually active will have HPV at some stage of their life,’ Harper says.  ’Normally there are no symptoms, and in 98 per cent of cases it clears itself.  But in those cases where it doesn’t, and isn’t treated, it can lead to pre-cancerous cells which may develop into cervical cancer”


The aforementioned bits are by themselves accurate; HPV infections are exceedingly common and in most cases they resolve by themselves, where the article takes those facts and mangles them up while taking up cudgels against the vaccine and being utterly clueless is in the interpretation that follows.


“Although these two vaccines are marketed as protection against cervical cancer, this claim is purely hypothetical.  Studies have proven “there is no demonstrated relationship between the condition being vaccinated for and the rare cancers that the vaccine might prevent, but it is marketed to do that nonetheless.  In fact, there is no actual evidence that the vaccine can prevent any cancer.  From the manufacturers own admissions, the vaccine only works on 4 strains out of 40 for a specific venereal disease that dies on its own in a relatively short period, so the chance of it actually helping an individual is about about the same as the chance of her being struck by a meteorite.””

Time to take a look at the individual claims and to look at the evidence.

[1] Although these two vaccines are marketed as protection against cervical cancer, this claim is purely hypothetical.  Studies have proven “there is no demonstrated relationship between the condition being vaccinated for and the rare cancers that the vaccine might prevent, but it is marketed to do that nonetheless.  In fact, there is no actual evidence that the vaccine can prevent any cancer.

Bollocks – there is evidence to show that it eliminates HPV infection and the consequent integration that is dependent on HPV infection to happen. The evidence associating HPV with cervical cancer, and HPV integration with cervical cancer, is extremely strong. All that is required is evidence that it can stop HPV infections, and that will cut cervical cancer incidence greatly. There is indeed very good evidence from a large trial showing that a vaccine that targets HPV16 can cut down HPV infection significantly AND also reduce the occurrence of precancerous HPV-associated lesions when they do happen. So the assertion that there is no evidence that the vaccine can prevent any cancer is completely turgid wibble.

Cervical cancers aren’t “rare” because HPV infection is so common, and the perceived rarity of cervical cancer in the United States referred to in the article is the result of a very good screening programme; women get their cervix screened for precancerous lesions regularly and precancerous lesions are burnt or frozen away, preventing tumour progression. Globally, cervical cancer is, after breast adenocarcinoma, the leading cause of cancer deaths in women (ref – )

I can’t see how you can stop HPV infections and not stop cervical cancer from developing, unless you decide to irradiate women’s cervixes to compensate for the lack of mutational sources other than HPV.

The reference to demonstrate that HPV is strongly associated with cervical cancer is here ( ). The figure is something like 98-99%, which, given how many people suffer and die from it, is massive.

From the manufacturers own admissions, the vaccine only works on 4 strains out of 40 for a specific venereal disease .


Elementary mistake – that there are 40 strains of HPV doesn’t mean that all of them occur at similar frequencies or that they are equally capable of causing cancers. Indeed, the five strains covered by one of the two available HPV vaccines account for 80% of the global cervical cancer burden, i.e, a very.large.number).


so the chance of it actually helping an individual is about about the same as the chance of her being struck by a meteorite.

More utter rubbish and sensationalistic journalistic nonsense. The chance of it actually helping an individual is the same as the chance someone will get cervical cancer from HPV infection, and the number of people who have to deal with cervical cancer is probably several orders of magnitude greater than the number of people being struck by meteorites.

Indeed, the article totally does not talk about HPV induced head and neck cancers ( ), which are associated with significant mortality and morbidity, and for which screening programmes do not exist. In other words, I’d always expect more due diligence from anyone writing about topics of interest for the general public who may be mislead by utterly dodgy arguments like the ones I’ve just quoted.



Building Brains: Symmetry, Synapses, and Shakepeare

This column will be about the brain, the gooey three pound jelly-like substance inside our skulls. Appearances can be deceptive, for this is quite possibly one of the most complex structures you will ever come across. All our memories, our knowledge, our hopes, dreams, aspirations, our beliefs, our likings, dislikings, our passions, our love, our hatred, almost everything that make us who we are are but activities in this lump of jelly. The billions of cells and trillions of connections that make up this structure are buzzing with activity throughout our lives. In fact, their activities are manifested as what we call Life. As one of my favourite bloggers put it, “”All life is here” in those tangled little fibres.”[1]. To understand life, we must understand the brain. In this column, I will try to pick up one fascinating story from neuroscience research every month and I will try and elaborate on it. My primary aim will be to present the research in a broader context and explain in the process why this matters in the bigger scheme of things, how the research is going to be useful in expanding our knowledge about how the brain (and, in a way, the universe) works. I will try and convey the sense of wonder and beauty that drives most of scientific research and I hope they will be contagious enough to inspire the reader to pursue science, if not as a career then at least as more than a passing interest.


This article is about how brains are built. The science that studies it is called Developmental Neurobiology and it is one of the most popular and active disciplines right now. Thousands of peer-reviewed articles are published each year[2]. So you can guess that it is impossible to give a flavour of the entire discipline in this one article. But every single discovery, every single article is actually fascinating in its own way and as is always the case in science, intricately intertwined with the bigger tapestry of our understanding. In this article, we will focus on one single aspect of neuroembryology (that’s another name of this subject) called Neurogenesis. As I mentioned in the introduction, I’ll try to pass on the enthusiasm that makes Developmental Neurobiology my favourite subject!

All of life starts from a single cell. From the humblest of unicellular creatures to the gigantic blue whale, each one of us started our lives as a single cell. So how is it that the single cell could divide itself in such an orchestrated manner to give rise to something so wonderful and complex? Shouldn’t all the daughter cells arising from that be identical copies of each other? How is this information regarding the fate of individual daughter cells transmitted? Turns out that the answer lies in asymmetry. If all the cell divisions, distribution of intracellular products and the extracellular environmental parameters were symmetrical, there would be absolutely no way to differentiate between the daughter cells. Symmetry has a lower information content than asymmetry. Once you develop a gradient of asymmetry, you can work on it and amplify it to regulate the flow of information in a very specific way. The initial bootstrapping through asymmetry is thus key to all of life.


Most of the studies in this field have been done in fruit flies, transparent nematode worms and vertebrates like xenopus and zebra-fish. But remarkably, the basic developmental processes in these organisms seem to be highly conserved throughout evolution. We have homologues of most of fundamental processes found in these simple creatures in the higher vertebrates. So although I will chiefly be discussing Drosophila research today, the conclusions we draw can be useful in understanding how the human nervous system is formed.


The initial asymmetry in Drosophila embryogenesis is established even before the fertilisation of the oocyte (the fruit-fly equivalent of egg) occurs. The nurse cells that surround the egg secrete substances that are imbibed by the oocyte and asymmetrically distributed in it. Among these substances are genes called bicoid (bcd) and oskar (osk) that establish the antero-posterior axis through their concentration gradients. Other genes called dorsal, cactus and toll establish the dorso-ventral polarity. Now after fertilisation the first genes that are expressed in the zygote (members of a class called the gap genes) arrange themselves in a pattern along this pre-established antero-posterior axis. We have the start of asymmetric life with coded spatial patterns. This anteroposterior patterning is then further reinforced by the expression of pair-rule genes, Hox genes and segment-polarity genes.


The development of the nervous system proper starts much later in the life cycle of an organism. But the same basic principle of asymmetric cell division plays a pivotal role in there as well.


The majority of functions of the nervous system are controlled at the most basic level by highly specialized cells called Neurons. Now, in order to generate the enormous diversity of function and connectivity in the nervous system, it is imperative that each neuron must be specialized to carry out a specific task. As a result, the neurons show tremendous variety in cellular structure, physiological functions, chemical properties and connectivity. Even the cells from a single region in the brain vary from each other in different aspects. For example, the granule cells of the cerebellum (the part of the brain that plays an important role in motor control) vary significantly in morphology and chemistry from the Purkinje cells of the same region. Similarly, the motor neurons, despite their structural and chemical similarities, have different molecular attributes that make them connect with specific muscle fibers resulting in the precision of movements that we see. This process of developing highly specialized neurons from comparatively similar precursor cells is called Neurogenesis. Question is, how is it done? What determines the fates of neurons?

Sydney Brenner once jokingly said that neurons are either European or American. The fate of an European neuron is mostly determined by its family lineage whereas that of an American neuron is largely shaped by the surroundings in which it grows up! As a general rule it is often said invertebrate neurons mostly belong to the former class whereas the ones in vertebrates fall in the later (any inference drawn herefrom is purely coincidental!). However, a close inspection tells us that the situation is not as binary as it looks. A cell is projected into a definite developmental trajectory by the environmental conditions that it is subjected to. So by default its daughter cells are only allowed to maneuver within that trajectory but with an enormous amount of variability (both reversible and irreversible) provided by the environment that they are now subjected to. More technically, this is controlled by two main processes called spatial patterning and temporal regulation of birth-dates. That is, the spatially coded patterns that surround a cell and the time of the final division that gives rise to it are most pivotal in determining its fate. These result in the cells expressing different transcription factors (regulatory proteins that cause expression of different genes) which ultimately determine their fates.

Spatial control of cell fate determination is all about making the right neurons at the right place. As we saw earlier, the Drosophila embryo is finely subdivided in the anterior to posterior axis into stripes of expressions of gap genes, pair-rule genes, Hox genes and segment-polarity genes. Now the neuroblasts (cells that eventually give rise to the neurons and all other classes of cells in the nervous system) are provided with intrinsic positional information by these genes. These anterio-posterior positional identity genes play important role in determining the identity of the neuroblasts.


The first of these spatially coded domains are initially specified by a gradient of the signaling molecule Sonic hedgehog (Shh). Now there’s an interesting story behind the nomenclature of this molecule. For those of you who were avid video-game fans in the 1990’s, must remember Sonic, the hedgehog. The gene was indeed named after the same character. The hedgehog genes were initially called so because mutations in them caused bristled appearances of the Drosophila larvae. The custom was to name the genes after different species of hedgehogs but when this gene was discovered, one of the postdocs in Clifford Tabin’s lab requested to name it after the popular Sega video-game character. Some had reservations about it but the naming was carried out anyway. There are criticisms for naming it thus, as mutations in the gene (actually, its homologue in humans) causes a serious condition known as holoprosencephaly. It seems rather cruel to name a gene after a video game character when mutations of it can cause such devastating conditions in children. But we are digressing from the main story. For a lively discussion of this through models and live demonstrations, watch the Howard Hughes Medical Institute Lecture on the basics of neuroembryology.


The progenitors of the neural tube are highly sensitive to the concentration of Shh, and this results in the graded expression of a group of transcription factors. We have a cascade of transcription factors being expressed in a directional manner being started by the initial asymmetry in concentration gradient like we mentioned earlier. Similarly there are other set of genes that divide the embryo and the nervous system along the dorsoventral axis. So you can imagine a sort of grid system being established where a neuroblast in any position can be uniquely identified by expression of these spatial coordinate markers of latitude and longitude. Genes specifying positional information along these two axes confer a positional identity to each of the neuroblasts in the developing nervous system. Once thus being expressed in a neuroblast, the spatial coordinate genes are inherited by all its progenies and they bear the indelible stamp of their birthplaces.

Having thus acquired their positional identities, each neuroblast divides asymmetrically to produce a copy of itself and a cell called the Ganglion Mother Cell (GMC). The neurobalst then goes on to divide further to give rise to a set of GMCs. But the really cool part is that each GMC can be identified not only by the positional information that it inherits from the neuroblast, but also from the order of its generation (that is, whether it was the first, second, or third GMC to be arising from a neuroblast). This temporal coding of information is carried out through a program of transcription factor expression. When the first GMCs are generated, the neuroblasts express a transcription factor called hunchback (hb). Later, they turn off the expression of this gene and turn on another one called Krueppel (kr). GMCs formed at the respective stages thus acquire these transcription factors. The expression of these transcription factors are linked to the cell cycle which functions as a kind of clock. Thus, in addition to the information about the place of their origins, the GMCs also carry with them information about the time of their birth. But it gets more interesting from here.


Typically, the progeny of a neuroblast inherits the temporal and spatial coordinates expressed by the parent at its time of birth. However, often the parent divides asymmetrically to give the intrinsic determinants to one of the daughter and not the other. How does a cell accomplish this feat of partitioning information asymmetrically among the daughter cells? The answer, it turns out, has a touch of Shakespeare to it!


Two factors, Numb and Prospero (Pro) play a pivotal role in the asymmetric distribution of determinants of cell identity. During the time of division, these tow factors are concentrated in the smaller daughter cell, the GMC, where Prospero enters the nucleus and determine the fate of the GMC. Numb, on the other hand, blocks a signaling pathway that renders the GMC free to move down the determination pathway. But how do Numb and Prospero get asymmetrically distributed in the cell in the first place? Two proteins called Inscuteable (Insc) and Bazooka (Baz) form a complex known as the Insc complex which binds to the apical membrane of the neuroblast and this complex causes the mitotic spindle (that’s like the apparatus that pulls different substances into the daughter cells during cell division) to be arranged vertically. In conjunction with the actin-based cytoskeleton (that’s like the transporter system of cells!) mechanism, this complex drives the distribution of several proteins along the vertical axis so that they are asymmetrically distributed. In particular, a cytoplasmic protein called Miranda is enriched at the basal neuroblast pole and it binds with Numb and Prospero to result in their asymmetric distribution. Wait a second! Miranda binds Prospero? What’s going on in here?


When I first read about these proteins, I was pleasantly surprised to find the Shakespeare reference. What on earth is Tempest doing in the middle of a Developmental Genetics textbook? I mailed  Prof. Chris Doe, the guy behind this fascinating nomenclature and asked him about the inspiration behind it. He said something wonderful, “You are right, they are from the Tempest:  Prospero the magician = the controller of fates!”[3] In a befitting tribute to The Bard, we have named the determiner of the fate of a neuroblast, which in a way is key to determining the fate of most of life, after the magician in The Tempest.


We are nearly at the end of our story. We have seen how information is passed on to the developing nervous system in a wonderfully coordinated manner. We have seen how neurons that will eventually define who we are become who they are. We have seen how their fates are determined by their origins and the environment they grow up in. We have seen temporal and spatial patterns giving rise to diversity in the nervous system. We have glimpsed into the most magnificent process in the universe where the most complicated computational device in the whole universe is being formed through self-assembly.


It has often been asked, what is the utility of studying this? We have our defences ready and we say that an understanding of neural cell fate determination will be important in understanding and treatment of neural diseases and injuries. In the future, we might be able to develop molecular therapies to repair damages in the nervous system. We might even be able to develop specific neurons from stem cells to use them in transplantation therapy.  More importantly, an understanding of this will be able to help us understand neurodevelopmental disorders like the autistic spectrum disorders better.


But I think there is a better reason to do all these. Science is in her uninhibited best when she is curiosity-driven. What better reason to pursue a career in research than to be able to name an all-powerful gene after the name of your favourite literary (or video-game) character? What better thing to study than the making of the mind? What better way to understand life than to watch it being formed? We don’t always to need to justify our passions in an utilitarian framework. Science is our most reliable probe into the nature of reality and pursuing it is, in my opinion, one of the best ways to spend the brief amount of time we spend on this planet.


Hope you will like this column. More interesting stories about the three pound jelly next month. Till then, have a great time!



1. From Neuroskeptic’s wonderful blogpost on the Morgellon’s disease:


2. About 25,000 articles were published on Developmental Neurobiology in the years between 2000 and 2004 (source: Development of the Nervous System by Sanes and Reh). We can only expect the number to be much higher than that in the last five years or so.


3. From the same email conversation with Prof. Doe, I also learnt that now there is a Caliban as well (PMID: 16103875). Shakespearem, it seems, is quite popular among developmental biologists!


Most of the other information in the article are from the following books:


Principles of Developmetal Genetics (edited by Sally A. Moody)

Development of the Nervous System (by Sanes and Reh)

Developmental Neurobiology (by Rao and Jacobson)

Principles of Neural Development (by Purves and Lichtman) (This book is currently out of print but you can download a copy for free from Purves’ website here)


A very short introduction to intratumour heterogeneity.

We have known for a while that tumours are basically unique and different, barring very few cancers with a very simple aetiology (like retinoblastoma).This led to the formulation of the concept of personalised cancer medicine wherein therapies would be tailored to match the mutational makeup of a patient’s tumour.

You would think that was a great idea until someone realised that tumour evolution isn’t a ladderlike series of clonal expansions as imagined before.

The original conception of tumour evolution as a linear process.

However, if you actually look at what evolution is like, there are multiple species that evolve in parallel, and researchers started to investigate if this was the case with tumour biology as well, in line with what the only diagram in Charles Darwin’s magnum opus; The Origin of Species.

Parallel Evolution

Studies have since gone on to look at what happens to tumour cells with time, and they do duly evolve and there are multiple subclones with allele frequencies that change in response to chemotherapy, for instance.


Tumours change with time in response to chemotherapy.

Using DNA sequencing methods, Tim Ley and coworkers showed that tumour evolution with time could follow either of two models – either one of the subclones acquired new mutations in response to chemotherapy and evolved to form the relapse clone or a previously present clone was driven by the selective pressure of chemotherapy to expand into the relapse population. (Reference – )

While this study only showed that there was a genetically distinct relapse population compared to pre-chemotherapy disease, it did not focus on parallel evolution. But Charlie Swanton’s group at the CRUK’s LRI and Tariq Enver’ s group at the UCL Cancer Institute did.

Enver and colleagues used a technique called FISH to examine changes in the copy number of candidate genes (gains or losses in how many copies of a gene there are) in Acute Lymphoid Leukaemia. They found that there were multiple subclones present at the same time with copy number changes that were independently acquired in some cases and were part of a complex, branched hierarchy. (Reference – )

a) represents assumed linear architecture. b) represents an inferred complex architecture using FISH with moderate complexity. c) represents evidence for an even more complex architecture with eight subclones from a different patient.

Charlie Swanton’s group used multiregion sequencing to explore heterogeneity in renal cell carcinoma and again found evidence for a complex pattern of evolution with multiple subclones evolving in parallel. (Reference – )

Self-explanatory. Charlie Swanton’s group found evidence for a complex, branched pattern of tumour evolution using an approach that combined deep sequencing with multiple biopsies of both the primary tumour and metastases.

And to make matters worse, we have emerging evidence for heterogeneity on an epigenetic level as well, and we might begin to have to consider the implications of that for tumour evolution. (Reference – ).

We are therefore now required to consider multiple biopsies for every case and then develop strategies to find what drives all these subclones, and then devise therapies accordingly. Things have gotten just a little bit more complicated for personalised cancer medicine…