Download Natural Salicylates and Cancer

18th February 2013
Natural Salicylates and Cancer
Summary of a discussion meeting.
Held in the All Nations Centre, adjacent to the University Hospital of Wales
On World Cancer Day: 4th February 2013
Present: Sir John Burn, Chair, Robyn Davies, Gary Duthie, Peter Elwood, Micaela Gal, John Gallacher,
Nick Gosman, Sam Hibbitt, Kerry Hood, Ruth Langley, Steve Lee, Chris McGuigan, Luis Mur, Dean Paul
Morgan, Mohammed Mustafa, Richard Neal, Andrew Omerod, Ifatm Parveeen, Stewart Sale, Monique
Simmons, Bob Steele, Martin Wiseman.
Apologies: Mansel Aylward, Ragib Ali, Les Baille, Steve Dennyer, Natasha deVere, Alexander Finlayson,
Ian Givens, Chris Hawkey, Alan Jackson, Malcolm Jackson, Howard Kynaston, Chris Butler, Peter
Rothwell, Philip Routledge, Will Steward, Sean Strain, Amanda Tristam, Terry Turner, Ian Young, Guy
Watson, Clare Wilkinson, Gareth Griffiths.
Professor Paul Morgan, Dean of Medicine in Cardiff University, opened the meeting.
Paul welcomed the visitors and commented on how the group is unusual in that it is
interdisciplinary, with representation of specialities other than medical: botanists, plant
physiologists, biologists, nutritionists, physicians, and the inevitable epidemiologist!
The topic of one particular salicylate, acetyle salicylic acid has been of interest in this
university for over 40 years, since Elwood and Cochrane reported the first randomised
evidence of a reduction in vascular mortality trial by aspirin. Now with its ability to
reduce the two major causes of death and disability, vascular disease and cancer this
particular salicylate is of enormous interest. The timing is therefore opportune for a
discussion of natural salicylates and their possible role in human health.
Croeso i Gaerdydd – Welcome to Cardiff!
John Burn: Introduced the topic of the meeting by stating that he has been fascinated by
salicylates for a long time. John told how, as a clinical geneticist, looking after families
with hereditary cancer, he had visited a family in which he noticed a young boy with
osteomata on his forehead. John knew that the lad would need to have his colon removed
before his twenty-first birthday and he wondered what could be done to avoid this or at
least slow down the development of colon polyps. He realised that preventive measures
against colon polyps and cancer could be tested efficiently and with small numbers in
subjects such as these.
John and colleagues therefore set up Concerted Action in Polyp Prevention or CAPP,
admitting an influence in the choice of the acronym that the cartoon character Andy Capp
had lived near where John had been born. The programme is still CAPP, but now,
Colorectal Adenoma/Carcinoma Prevention Programme. CAPP 1 failed to give evidence
of protection from aspirin. John and colleagues then enlisted patients with Lynch
syndrome, in which there is an error in the clamp system which identifies and repairs the
‘spelling mistakes’ in DNA. Fifty to sixty percent of these subjects get cancer in their
early 40s. 1,000 gene carriers were recruited, and after two years the answer was – the
aspirin made no difference whatever! After four years of aspirin however there was a
clear difference in cancer incidence with a hazard ratio of 0.60. A further analysis looking
at the number of cancers, rather than the number of patients with cancer, showed that
after about four years prophylaxis there was an HR of 0.45 (95% CI 0.26, 0,79) and an
Incident Rate Ratio of 0.42 (0.25, 0.72).
John then talked about the possible mechanisms of aspirin, and he described how he had
met colleagues in Leicester and learnt how salicylates can act as a trigger for apoptosis in
plants. This prompted him to consider whether natural salicylates are something that we
used to get in our diets but are lost now with modern farming methods. Maybe therefore
aspirin prophylaxis is putting back something we had been evolved to expect in our diet.
CAPP 3 is now planned. This will test 100mg, 300mg and 600mg. and will give
evidence on side effects. John pointed out that deaths from colonoscopy (due to
perforation) are more likely than deaths attributable to aspirin! There are probably about
60K gene carriers for hereditary bowel cancer, but only about 6K are known. In a
commercial venture John has developed a device that will extract, amplify and analyse
DNA rapidly and cheaply, and will facilitate the identification of gene carriers.
Peter Elwood: Aspirin, salicylates and cancer. Peter presented a background to the
present gathering. Although the history of salicylates goes back to Hippocrates and
beyond, and includes the Rev Edward Stone, the production of acetyl salicylic acid by
either Hoffman or Eichengrun, in the dye factory of Fredrich Bayer, led to the focussing
of interest on aspirin, rather than salicylates. Then in 1974, the publication of the first
randomised trial showing a reduction in deaths from myocardial infarction stimulated a
huge interest across the world in the drug.
Around that time, in 1974 I had contact with Stan Pierpoint, a botanist, knowledgeable
about salicylates. His view was that the medical properties of aspirin in humans might
simply be a logical consequence of their role in plants, and he urged me to test aspirin
against cancer. I search the literature and found only a little work in animals suggesting a
reduction in cancer metastases by aspirin. In 1988 however, the cohort study in
Melbourne was published by Kune and colleagues, showing a negative association
between aspirin taking and incident cancer.
In 2005, I made contact with Gary Duthie, Luis Mur and others, suggesting that we
review the evidence from botanical, animal and other sources for an effect of aspirin and
salicylates on cancer. In 2009 we published a review in the Lancet, and in addition to
evidence from botanical and animal sources we included summaries of Mendellian
randomisation studies and a host of observational case-control and prospective studies in
human subjects. At the same time we set up a Citizens’ Jury in an attempt to see how the
general public would judge the evidence available at that time.
In our review paper we stated that because of its benefits in vascular disease, it would be
unacceptable, and probably unethical for randomised trials of aspirin to be conducted,
and decisions about aspirin prophylaxis of cancer would therefore have to be based on
evidence other than randomised trials.
How wrong we were! Within a year, Peter Rothwell and colleagues began to report
evidence from long-term randomised trials showing a reduction in cancer in the subjects
who, up to 20 years previously, had been randomised to aspirin. The remarkable ad hoc
randomised trial of John Burn was also published at this time. However, I believe that
there are two further pieces of evidences in these trials that strengthen the case for aspirin
prophylaxis. A number of papers published around 2003, including one from this
University, had shown that aspirin added to cell cultures enhanced the cellular DNA mismatch repair process, and this had led to predictions of benefit from aspirin. The patients
in John Burn’s trial suffered from a genetic error in mismatch repair. Then further, a
number of papers from 1995 and later, surmised that as the protection from aspirin
appeared to be at a cellular level early in carcinogenesis there would be a delay of
perhaps 5-10 years before any possible benefit from aspirin could be detected clinically.
This prediction was also fulfilled in the reported trials.
The bottom line in this situation is of course whether the benefits of low-dose aspirin
outweigh the risks. The trouble in this is that while cardiologists restrict their evaluation
to vascular benefits alone, oncologists restrict theirs to cancer alone. What is needed is
an evaluation of the total benefits – vascular plus cancer – against the risks. And such an
evaluation has been reported! Thus, Jacobs and Patrono, three powerful, but very
cautious investigators have published an evaluation and their conclusion is that: “Even a
10% reduction in overall cancer incidence beginning during the first 10 years of
treatment [with low – dose aspirin] could tip the balance of benefits and risks favourably
in average-risk populations.” (Nat Rev Clin Oncol 2012;9:259-67). Now however,
evidence of benefit from the treatment of cancer by aspirin is accumulating. (See the talk
by Ruth Langley later).
The side effects of aspirin however are serious and lead to reluctance to recommend
aspirin by doctors and others. A stomach bleed is a crisis, yet there is no evidence from
either clinical trials, or community based studies, that fatal bleeds are any more common
in subjects taking aspirin than in the general community. Cerebral bleeds are very much
more serious, but there is evidence, albeit very limited, that suggests that cerebral bleeds
are much less likely if hypertension, if present, is adequately treated.
Predicting the future is hazardous, yet the incidence of vascular diseases is falling rapidly
and the use of aspirin in vascular protection is becoming less and less important. Cancer
is however likely to increase, and as Rothwell predicts, aspirin is likely to become of
increasing importance in cancer prevention.
But why aspirin? Why not natural salicylates, or aspirin plus salicylates? It is to try to
answer this question that this meeting had been arranged!
Stewart Sale: Curcumin as a cancer preventive. In Leicester there is a large group
examining amongst other things, dietary factors of possible relevance to cancer. Clues
are looked for in the diet, compounds are isolated, analogues synthesised, in vitro tests
conducted and then, if favourable, pilot studies are conducted in human subjects. To
show promise, compounds have to have high efficiency, low toxicity, known mechanisms
of action, acceptability of taste, taken orally and be low cost.
Much work is focusing on the curcumin, a polyphenol from tumeric. It has been
consumed for about 4K years and about 1.5g are included the average Asian diet and it is
used extensively as a medicine throughout Asia. In India large and small bowel cancers
are about 10-15% lower than in the UK, and in Leicester colorectal cancer – even in third
generation Asian subjects the incidence is reduced by about 20% to 80% of the nonAsian population.
Preliminary testing shows that it has anti-proliferating activity, it scavenges free radicals,
it inhibits cyclooxygenase; it induces both apoptosis and angiogenesis and inhibits
protein kinase C. Much testing has been done in a mini-mouse model, and 0.2 and 0.5%
levels lead to a clear reduction in polopi in the small intestine, but no clear effect in the
colon. Cox2 levels in the gut mucosa are however reduced by about 60%.
Pharmacokinetic studies show a very low availability with only traces in the plasma – yet
some is detected in the mucosa and the liver. 0.2 and 0.5% doses are required in the
mouse to elicit effects and this suggests about 1.6g/day in humans.
Pilot studies are in progress in humans to study pharmokinetic properties, to obtain a
guide to dosage. Further testing will examine safety and will seek surrogate markers of
efficacy in order to indicate likely efficacy before long-term trials have been completed.
Garry Duthie: Uptake of dietary salicylates from food. Garry paid tribute to John
Paterson, who did a lot of work on salicylates until his death a few years ago. The main
questions Paterson and Garry had posed were: what are the main food sources of
salicylates, how rich is the diet and how likely is it that nutritional amounts might prevent
pathogenic events, and if they do, how is this achieved?
The content of individual foods in the western diet are relatively low and the
consumption of 1Kg of pasty would yield about 5mg salicylate, 1Kg of spaghetti bol.
would yield 3mg and 1Kg of banana about 0.4mg. Sources in the average Scottish diet
are alcoholic beverages (20%), other beverages (13%), herbs and spices (17%) tomatoes
12% giving a total intake of about 4mg/day. Fruit and vegetables contribute only about
16% and 9% respectively. Indian communities in Scotland have however a much higher
intake, at about 12 mg/day, herbs and spices contributing about 38%, vegetables about
18% and fruit about 13%.
Turning to subgroups of the population Garry gave estimates, based on urinary excretion
of salicyluric acid: omnivores (0.07), vegetarians (0.11), South Indians (0.26). While the
range around these levels overlap with levels in subjects taking low-dose aspirin, there is
a difference of an order of magnitude, with aspirin takers having levels about 10 fold
greater. Nevertheless, levels other than those from aspirin, could still be bioactive and
we should therefore be prepared to look for effects at micromolar concentrations.
The half life of salicylates in blood is about 2-4 hours. There appears however to be large
inter-person variability in absorption from the food matrix and in a small study of three
volunteers only one excreted detectable amounts of salicyluric acid after a dose of
salicylates.
Garry then urged that phenolics in general should be studied. Salicylates are only one of
a family of phenolics, many of which have bioactive functions, inhibiting prostaglandins
and mopping up free radicals etc. Total daily intake of dietary phenolics may exceed
600mg and the bioavailability of some of the cinnamic and benzoic acids appear to be
high.
The literature, to which Gary has himself made important contributions, suggests that, in
Scotland, average intakes estimated from studies over several months are about 3.5-4mg
/day. Poor absorption and rapid excretion of some, including salicylates might be
expected because after all plants evolved phytochemicals such as salicylates to deter
animals from eating them!
Luis Mur and Ifat Parveen: Salicylates in plants: an unsuspected nutraceutical?
Luis commenced by pointing out that plants are enormously powerful factories producing
more than 100K metabolites. In Aberystwyth they have a huge metabolomic facility and
take on large scale metabolic profiling, indentifying amongst many things, possible
natural products with microbiological properties.
Salicylic acid and other polyphenols give protection against animal consumption, they
confer disease resistance, and they respond to any stress, whether biotic, or
environmental, such as heat or cold. Plant species which are grown for the human food
chain are however usually selected to have low salicylate levels, to ensure acceptability
through cost and palatability. Furthermore, when plants secrete salicylate they can
become disfigured and unacceptable in the market place.
Infection leads to a very rapid response by a plant, with salicylate being secreted within a
few hours, and levels remain high for perhaps a week, tending to make the plant
unattractive for consumption. Plants which have been exposed to cold (5C) or heat (38C)
for a few hours also have high levels, lasting perhaps a week.
There is an interaction with cell mitrochondria in plants.
A new investigation in Aberystwyth focuses on milk. The first stomach in the cow, the
rumen, is full of bacteria, fungi and protozoa, and salicylate is produced. The type of
grass grazed by the cow appears to be relevant to this salicylate production in the rumen.
Whether or not this salicylate gets into the milk is now being investigated. But even if it
does, questions will arise about the effects of pasteurization and storage etc.
In summary, plants are a source of many nutraceuticals; salicylates are a response to
stress and plants which have been cosseted while growing can have very little salicylate.
Dairy produce may be an important source of salicylates.
Martin Wiseman: Vegetables, fruit and cancer. In his talk Martin commented
repeatedly on the changes which have taken place over the past twenty and more years in
the evaluation of nutritional evidence. In 1997 the World Cancer Research Fund
published an evaluation of foods and cancer. Almost all the evidence came from
observational studies with some work on mechanisms. The bottom line was highly
favourable and the recommendation to eat more fruit and vegetables, led eventually to the
well known phrase: ‘Five a Day’! Since then, the consumption of fruit and vegetables
has increased, probably mostly through an increase in processed items.
That 1997 report claimed that much evidence supportive of cancer prevention. Martin’s
work has however been with a report published by the same body in 2007. This was
based on a series of systematic reviews by independent panels, reviewed by a central
committee, and it led to what is one of the most authoritative reports on the topic of
foods and cancer. The evidence is still observational and mechanistic, with
understandably, evidence from only a very few clinical trials.
The 2007 report made 10 recommendations, only a few of which relate directly to plant
foods. The first three are about body fatness, physical fitness and foods that promote
weight gain. The last two relate to special situations, including pregnancy. Plant foods
are dealt with very broadly in an attempt to encourage people not to think of individual
foods as possible ‘magic bullets’, but rather to consider food, in its relevance to health, as
a ‘package’.
Martin then reviewed the 2007 report, showing various tables summarising evidence
from case-control and prospective studies. On the whole, evidence from case-control
studies is noticeably more favourable than that from prospective studies, but suffer more
from imprecision and bias, the latter not only in the reporting by subjects, but also from
the likely publication of studies with positive results.
In the 2007 report, individual foods were grouped into those for which the evidence of
benefit was judged to be ‘convincing’, ‘probable’ and that which failed to reach either.
For no food was the evidence judged to be convincing. Foods judged to be ‘probably’
protective against cancer included non-starchy vegetables, items containing fibre, garlic,
fruit, folate, leucopene and/or selenium. For none of these however does the 2007 report
judge the evidence to be of the ‘highest’ level – in contrast to the earlier 1995 report. The
strongest evidence suggestive of cancer reduction relates to upper aerodigestive cancers,
including oesophagus and stomach, but in these cancers exogenous carcinogens may be
important.
So, in summary, evidence for an inverse association between plant foods and cancer is
less convincing than believed by most people, and the search for a diet that would protect
against cancer has changed to an aim for a ‘nutritional state’ that is consistent with health.
An agreed important aspect of a plant based diet is a likely reduction in the energy
density of such diets.
Monique Simmons: Work in Kew Gardens. In Kew there are about 200 scientists,
about 75 of which are taxonomists, looking at relationships between plants. Monique
believes that these have a lot to offer in the work being discussed by this group.
Since 1985 work on the profiling of plants has been done and there are now large
libraries on the diversity of plant compounds, listing about twenty thousand different
species. This is done for taxonomic reasons, but also in a search for novelty. Some of
the pharmaceutical firms were interested in this, but the approach did not prove
profitable. There has therefore been a move away and the emphasis now is on looking at
the role of compounds within plants – interactions with herbivores, pathogens etc., and
thus, exploring traditional knowledge about plants.
This last avenue of enquiry has been much more profitable. For example: the hypothesis
that compounds in plants resistant to insects might have anti-cancer activity led us to
explore rice, and this led to Tricin, a small polyphenol. Work therefore focused on Tricin
and other polyphenols in rice. These compounds are however removed by processing,
but rice is of course a grass, and we have found grass to be loaded with the same
compounds!
Monique recommended that phenolics other than salicylates should be looked at and she
told of a search conducted for purposes very different to medical, namely cosmetics.
Starting with the Salix species they searched for salicylates and found the richest source
to be in broad beans… a plant of no interest to the cosmetic industry(!) but of possible
relevance to today’s discussions.
Finally, Monique appealed for more interdisciplinary collaboration, particularly from
medics!
Nick Gosman: Ways of increasing salicylate levels in plants. Based on experience
from two ongoing projects in the Novel and non-Food Crops Department at NIAB, Nick
presented a talk describing how plant breeding approaches might be used to increase
dietary salicylate levels. The Novel and Non-Food Crops team focus on the development
of plant-based sources of products in biopharmaceuticals, nutraceuticals and other aspects
of nutrition, biomaterials and some types of fuel. Two projects have specifically focussed
on the development of natural sources of two medically active compounds, artemisinin
from Artimesia annua and stearidonic acid from Buglossoides arvensis.
The breeding strategy used aims to simultaneously select improved parental lines from
genebank collections & develop improved cultivars from crossing programmes. Three
complementary approaches have been successful in developing commercial cultivars
with improved agronomic and extraction performance. In the first, line improvement of
genebank accessions identified individual plant lines with suitable agronomy for UK
conditions and increased levels of extractable compound. Selected lines were used in a
hybridization programme to develop segregating populations that were again selected on
a plant-by-plant basis. The best progeny lines were fed back into field trials to be
compared with the original newly acquired accessions. The above scheme is, however,
dependent on the existence of substantial genetic variation for the traits under selection.
Aremesia annua has been used in traditional Chinese medicine for hundreds of years. The
efficacy as a prophylactic anti-malarial of tea prepared from A. annua leaves is
questionable. However, modern artemisinin-based combination drugs, trade names
Coartem and Riamet, have been shown to be highly effective in the prevention and
treatment of malaria.
Artimesia germplasm collected by NIAB has undergone rigorous field trials to select for
lines with improved agronomic and artemisinin extraction performance. Germplasm has
been collected from genebanks and private collections. However, it is already a crop in
China and Vietnam, but commercial cultivars tend to be low yielding for artemisinin.
Therefore, wild accessions were selected for better germination and establishment under
UK conditions. Agronomic trials were performed to identify optimum weed control, plant
spacing and harvest timing.
Results obtained at the end of the original three year project in 2009 were promising.
Compared to control (an average of five standard accessions identified at beginning of
project) selected lines out-yielded the standards (that yield <1.6% of artimesinin by
weight of leaf material) by between 10 and 20%, potentially significantly increasing the
yield per hectare. Parental selections all show improved yield of artemisinin compared to
the controls after three years of selection for improved field and extraction efficiency
performance. All high-yielding accessions were used in a diallele crossing programme to
generate populations for field assessment.
The percentage of artimesinin in the leaf during growing increases over time with a peak
in September (harvest). In 2009, trials indicated that the NIAB project had achieved an
increase of >1% after 3 years selective breeding. These results demonstrate that relatively
rapid progress can be achieved in an unimproved plant species. However, Chinese
produces can make money on 0.2%, but with higher input costs, this return is not
economically viable in the UK because the world price tends to be low. But high yielding
lines identified in the NIAB project are now commercialised abroad in Madagascar &
China; a classic case of ‘exporting coals to Newcastle’! Many of the lines developed outperform lines developed by the CNAP project.
The Buglossoides example: Stearidonic acid (SDA) is a key precursor in the biosynthesis
of long chain (LC) omega-3 (or n-3) polyunsaturated fatty acids (PUFAs) such as
eicosapentaenoic acid (EPA, C20:5n-3) and Docosahexaenoic acid (DHA, (22:6 n-3) that
are commonly found in fish oils. The potential health benefits of increasing n-3 PUFA in
an ageing population with low levels of fish consumption and declining availability
highlights the need to develop alternative dietary sources of these fatty acids. The only n3 LCPUFAs common in plants are ALA (found in flaxseed oil) and SDA (found in
blackcurrant seed oil and Echium plantagineum seed oil). The SDA content of
blackcurrant seed oil is low (~3%) and Echium is a very low yielding plant species.
Buglossoides arvensis (syn Lithospermum arvensis, or Corn Gromwell) is potentially a
superior source of SDA with major yield/agronomic and hence cost advantages over
Echium plantageneum. It should be noted here that the health benefits of SDA (18
carbon chain fatty acid) have been brought into question by recent research that suggests
that only longer chain fatty acids found in fish oil offer proven health benefits; a point
raised by Martin Wiseman at the meeting.
SDA extracted from the seeds. The NIAB Novel and non-food crops group, NIAB-TAG
agronomy research department and Technology Crops have developed a complete
agronomy package for this new crop. This package minimises the risk to growers
attempting to grow the crop for the first time. We have completed trials for seed rates,
planting dates, nitrogen rates, regional trials, herbicides and fungicides. Accessions were
collected from word genebanks, including Kew Gardens. The project also identified
potentially higher-yielding winter types from central Europe (centre of diversity) that
need cold treatment; vernalization (usually all spring). SDA analysis was done at NIAB
using HPLC (calculate % of oil that is SDA). There was very narrow variation for traits
of agronomic interest, therefore, mutant screening to induce variation for traits that are
not available naturally, e.g. herbicide tolerance and higher oil content.
Herbicide tolerance breeding programme: Within a commercial cropping system,
tolerance to herbicides is critical for improving establishment and early growth of
seedlings under pressure from common broad-leaved weeds. EMS mutagenetisis of seed
followed by selection of surviving seedlings in the field after being exposed to several
common herbicides has identified germplasm that is viable under standard herbicide
regimes. Populations of approximately 10,000 mutagenized plants were grown out and
selfed to M2 for field herbicide selection. From each population, single plants were
planted in a glasshouse and allowed to self pollinate for two generations post mutagenesis
(M2). Tolerance to commonly-used herbicides is crucial for commercial production
systems. Variation for herbicide tolerance was created by EMS mutagenization. Selection
on M2 plants in the field <1% of plants carried mutation(s) that conferred herbicide
tolerance.
SDA and overall oil content of seed of winter sown accessions was greater than that of
spring sown. Many spring lines were not winter hardy, but some could be also be grown
in the winter and required some cold treatment before they would flower (vernalization).
SDA production of the best lines was found to approach that of GM soybeans. Now
several lines are being commercialised in UK by Technology Crops & NIAB.
Increased sources of dietary salicylates: Traditional sources of salicylates include the
bark of willow (Salix), mentioned by Hippocrates in the 5th century (where the bark
extract, salicylin gets its name). Preparations have also been made from meadowsweet,
but early extracts were toxic. Dietary sources of salicylic acid are low, in the range 0-5mg
per 100g, however, high levels are reported in spices such as Cumin in range of 45mg per
100g? In Indian communities where large quantities of spices are consumed (rich in
salicylates), the incidence of colorectal cancer are the lowest in the world. In western
countries, where consumption of spices is considerably lower, increasing levels of dietary
salicylates in common foodstuffs would be beneficial, e.g. even a small rise in salicylate
level in a staple could make a significant increase in dietary intake.
The tomato example: Increased levels of salicylates can be achieved by subjecting plants,
including tomato, to abiotoc stresses such as drought, so manipulation of the environment
would potentially amplify increased baseline levels (a straightforward option in a mostly
glasshouse grown crop). Plenty of genomic tools are available, especially genetic maps,
mapping populations, molecular markers, etc. If naturally occurring genetic variation for
salicylates in tomato turned out to be low, screening for induced mutations, e.g.
TILLING would be a good option since it does not involve genetic modification.
As in all domesticated crop species, genetic variation in cultivated tomato tends to be low
due to domestication and selective breeding. However, I have not found specific
references to levels of variation for salicylate levels among cultivars. Wild species of
Solanum provide greater genetic variation, but crosses to such germplasm require many
generations of selection to recover varieties with the cultivation and flavour traits
required. Having gene or molecular markers helps during backcrossing though.
TILLING methodology: The reverse genetic technique of TILLING allows for the
identification of an allelic series of mutants with a range of modified functions for desired
genes. DNA from a collection of EMS-mutagenized plants is pooled, subjected to PCR
amplification, and screened for mutations using denaturing HPLC (DHPLC). DHPLC
detects mismatches in heteroduplexes created by melting and annealing of heteroallelic
DNA. Among the lesions detected are base transitions causing miss-sense and non-sense
changes that can be used for phenotypic analyses. TILLING populations in tomatoes are
relatively easy to establish and there are many freely available within the tomato research
community, e.g. The Micro-Tom TILLING platform illustrated in the slide presentation.
In summary: For unimproved, wild species, considerable work is needed to develop a
crop for agricultural production. However, rapid improvement in extractable quantities of
target substances can be achieved where there are high levels of genetic variation and a
rapid method for screening. Increased intake of salicylate in Western diets could be
achieved by developing cultivars of existing crop species with increased levels either
through selective breeding or targeted mutation, or both.
Ruth Langley: The Add-Aspirin trial. Ruth commenced by posing the question; why
have oncologists been so slow to evaluate aspirin as a treatment for cancer? She pointed
out that oncologists require a substantial basis of evidence before a new treatment can be
introduced, and new drugs are usually first tested in patients with advanced metastatic
spread with response rates (substantial reductions in tumour size) as the measure of
activity. Aspirin would not pass this test, but she reflected that the current interest in
aspirin as an anti-cancer drug may change the way that oncologists view the current
methods for evaluating potential new anti-cancer drugs.
There are several clinical sources of evidence confirming the anti-cancer effects of
aspirin . First, the importance of the CAPP2 trial cannot be over-estimated the first
positive ad hoc randomised trial, with cancer reduction as the primary outcome measure.
Secondly, Peter Rothwell’s studies which show a reduction in incidence and mortality
across cancer types. Furthermore, the reduction begins relatively early suggesting a role
for aspirin in the treatment of cancer, supported by a reduction in metastatic spread.
Further to all this, evidence of benefit from aspirin after primary therapy is accumulating
from observational studies in a range of cancers: breast, gastro-oesophageal, colorectal
and prostate.
There is therefore opportunity for a randomised trial and it is likely that benefit will be
best shown immediately following primary radical therapy (surgery and/or chemotherapy
and/or radiotherapy), with aspirin used as an additional adjunct treatment. A major trial
is therefore planned. There will be an overarching protocol with four separately powered
randomised phase III trials: participants with colorectal, breast, gastro-oesophageal and
prostate cancer will be included and randomised between, aspirin 100mg daily, 300mg
daily and placebo for at least 5 years. It will be conducted in India and the UK, because
the benefits, if confirmed, could be of particular importance to poor-resource
communities. Approximately 2,500 patients will be included in each tumour-specific
trial. Secondary outcomes will include overall survival and toxicity.
Ruth concluded with a brief reference to a paper of an observational study in which only
patients with tumours expressing a mutant gene, PIK3CA, appeared to respond to aspirin
(Liao et al NEJM 2012), suggesting that it may be possible to identify patients especially
suitable for aspirin.
John Gallacher: Web-based epidemiology. John is a member of the main Steering
Committee for BIOBANK, the prospective study based on 500,000 subjects throughout
Great Britain. Working with this study gave John ideas about large scale studies which
would avoid face-to-face contact with subjects, thus reducing costs enormously. Studies
based on the web, with remote recruitment, remote assessment and remote follow-up
would fulfil these criteria – if feasible. The strategy has proved possible, yielding, as it
were, a living laboratory within which the whole range of epidemiological aims can be
achieved. In his talk John used the word ‘Fungible’ a number of times, which he later
defined is as: the trading of benefits!
John has completed a pilot study using this strategy. 15K older people were mailed and
invited to register on the web. 663 were recruited, estimated to be 11% of those
connected to the web – a higher response than had been achieved by BIOBANK! 75% of
the respondents completed all the modules in a long questionnaire taking 60 minutes to
complete. One third of the respondents were then asked to submit a dry blood sample on
filter paper, and 70% complied, while another third were asked to submit a buccal smear
and 75% complied. Following the collection of these data and samples, record linkage
was achieved for 99% of the subjects.
Of course the respondents were selected, but what really matters is whether or not the full
range of each variate is represented, and this was achieved in age, and in an index of
deprivation. The main data collected related to cognitive function, and psychological
state, and for each of these measures the distributions of results were compared with the
distributions which had been obtained in studies using the same tests in face-to-face
interviews. The distributions were very closely similar – even odd querks in the data
were similar in the two sets!
All the components required for large scale epidemiological studies can therefore be
obtained remotely, at low cost, and can be successfully lined with medical records. For a
number of reasons, including the existence of the NHS, web-based studies, could be done
better in the UK than almost any other country. Furthermore, there is little doubt that
studies relevant to today’s topic – the relevance of diet, and salicylate intakes in
particular, to cancer incidence - would be feasible in web-based studies in the UK.
SUMMING UP: Martin Wiseman. Martin commenced by reviewing very briefly how
salicylates, in common with many other items, had been of great interest but had then
been forgotten, eclipsed by another interest – in this case, aspirin. Aspirin has been the
focus of intense activity, and rigorous research studies have sorted out the risks and the
benefits in vascular use, and now its uses in cancer are being rigorously investigated.
There have been two interlocking strands in the discussions today: academic type studies
of diet and cancer, and secondly, using the knowledge gained from these, attempts to
develop drugs or nutraceuticals for either prevention of treatment of cancer. Certainly a
variety of approaches within both of these is required, ensuring a broad evaluation.
A number of issues have been raised. The story begins with observations that people
with a plant-based nutrition have a low risk of cancer. There are difficulties in
characterising this and in particular in identifying possible single foods or nutrients.
Difficulties include imprecision because of the huge variability in dietary intake and
confounding between food items, variability in the content of any particular item within
food items, dependent upon season, storage, preparation etc, all affecting the content of
any nutrient of interest. Furthermore, people vary in their absorption and in their
metabolism. In fact, we probably have been too influence by aspirin, which appears to
work at mg levels. Perhaps we should be considering possible effects at micromolar
levels, at which powerful cellular effects can occur.
Then we need to identify biomarkers of exposure, additional to urine and blood levels,
and spot blood samples could be useful in the monitoring of remote cohorts. But
biomarkers of beneficial effects are required, such as the effect of salicylates on
angiogenesis, as mentioned by John Burn.
Milk and dairy foods seem to be of particular interest as sources of salicylates, as
mentioned by Luis, and alcoholic beverages, tomatoes need to be examined further.
This, Martin concluded, is what I got out of the day’s discussions – enough for several
decades of research!
Micaela Gal, Mohammed Mustafa and Robyn Davies - Rapporteurs