# New Pathways for Alimentary Mucositis

Authors: Joanne M. Bowen; Dorothy M. K. Keefe
Journal: Journal of Oncology
 (2008)

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## Abstract

Alimentary mucositis is a major dose-limiting toxicity associated with anticancer treatment. It is responsible for reducing patient quality of life and represents a significant economic burden in oncology. The pathobiology of alimentary mucositis is extremely complex, and an increased understanding of mechanisms and pathway interactions is required to rationally design improved therapies. This review describes the latest advances in defining mechanisms of alimentary mucositis pathobiology in the context of pathway activation. It focuses particularly on the recent genome-wide analyses of regimen-related mucosal injury and the identification of specific regulatory pathways implicated in mucositis development. This review also discusses the currently known alimentary mucositis risk factors and the development of novel treatments. Suggestions for future research directions have been raised.


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## Body

## 1. Introduction

Recent research has indicated that
cytotoxic chemotherapy causes unwanted normal tissue damage via its effects on
a multitude of cellular regulatory pathways [1, 2]. In particular,
investigations have begun to elucidate the role of regulatory pathway
activation and suppression in regimen-related gastrointestinal toxicity [2, 3]. Mucosal toxicity
following anticancer treatment is clinically referred to as mucositis.
Mucositis was once separated into oral and gastrointestinal, however it is now
widely known as alimentary mucositis, referring to the damage which occurs
along the entire orodigestive tract. Alimentary mucositis affects the mucosa,
causing mouth and throat pain, ulceration, abdominal pain, bloating, vomiting,
and diarrhoea depending on the target tissue [4–6]. The general mechanisms underpinning the
development of mucositis are thought to be the same regardless of the location
along the length of the tract. However, the kinetics of symptom development is different in each
region, which is thought to reflect the local turnover rate of epithelial cells
and specialised differentiation for local function [4, 7]. The frequency of alimentary mucositis varies
depending on the cancer and treatment, ranging from 10–40% in patients
undergoing standard chemotherapy for solid tumours, up to 60–100% in those
undergoing high dose conditioning chemotherapy for stem cell transplantation [5, 8].


## 2. Burden of Alimentary Mucositis

Alimentary mucositis represents a
significant clinical and economic burden in oncology. The presence of any mucositis
during a cycle of chemotherapy significantly increases the risk of dose
reduction, the frequency of infections and bleeding, and increases the length
and cost of hospitalisation. Reduction in treatment doses leads to reduced survival [9], and mucositis is also a risk factor for mortality due to
its association with infection [10]. What is more, severe
mucositis has been shown to be a significant risk factor for inferior overall
survival, relapse mortality, and nonrelapse mortality in specific settings [11]. Resource utilisation for
patients during episodes of mucositis is also significantly increased, with the
need for nutritional adjuncts including fluid replacement, liquid diets, and
total parenteral nutrition. Due to the association with infection, antibiotic
therapy is also more common in patients with mucositis. Combined, this
translates to an incremental cost increase of US$3500 per cycle of standard
dose chemotherapy with oral mucositis [12, 13] and US$6000 with both
oral and GI mucositis [14]. While the full burden of
mucositis has yet to be defined, a prospective study is ongoing to determine
its true quality of life and economic impact.


## 3. Current Overview Mucositis Pathobiology

Accumulating
evidence has strengthened the proposed biological model of mucositis [15, 16]. It is
known as the multiple mechanism model, as it represents a divergence from the
initial linear view that mucositis was purely a result of cytotoxic
agent-induced damage to basal epithelial cells [17, 18]. There is
indeed interference with epithelial stem cell turnover and increased apoptosis
following anticancer treatment, however this is but one component of the
pathobiology of alimentary mucositis. Rather, the events that lead to mucosal
injury are multifactorial, complex, and pan tissue and are driven initially by
the submucosa via endothelial signalling [16, 19].
Epithelial breakdown represents the clinical stage only and is associated with
loss of barrier integrity, host infection, and considerable pain [4, 7, 20–22]. Mucositis
has been described as having 5 phases, which are overlapping and interactive [16, 22]. The 5
phases are initiation, primary damage response/upregulation and message generation, signal
amplification, ulceration, and healing. Briefly, cytotoxic agents initiate
damage through the generation of reactive oxygen species which causes both
direct damage to tissue components of the mucosa and also activates secondary
signalling. The primary event of the message generation phase centres around
activation of the transcription factor NFkB, which leads to the upregulation of
many genes involved in perpetuating mucosal injury, including proinflammatory
cytokines, adhesion molecules, and cyclooxygenase-2.  During the third phase, a feedback loop occurs
whereby the proinflammatory cytokine, TNF, acts on a number of pathways to
reinforce NFkB activation and the ceramide pathway. The ulcerative phase comprises
loss of mucosal integrity and bacterial colonisation, with subsequent further
proinflammatory cytokine production. Alimentary mucositis is usually
self-resolving once treatment ceases, and healing occurs with renewal of
epithelial proliferation and differentiation and reestablishment of the normal
local microbial flora [16, 23, 24]. It is evident that this current model incorporates
sequential interaction between all cell and tissue types of the mucosa and
submucosa, as well as tissue factors, cytokines, and elements of the luminal
environment [25, 26]. Among others, the
pathways implicated in this model include ceramide signalling, extracellular
matrix turnover, oxidative stress signalling, apoptosis, cytokine signalling,
and cell cycling.


## 4. Special Considerations for the Git

Mucositis
pathobiology has been extrapolated mainly from research in the oral
setting. However there is rapidly
accumulating evidence that supports the theory that mechanisms are similar
along the entire length of the alimentary canal. In models of mucositis, inflammatory
mediators, TNF and NFkB, have been shown to increase along the length of the
gastrointestinal tract corresponding to histological damage [27, 28].
Furthermore, proinflammatory cytokines, interleukin 1 and 6, are increased
following methotrexate treatment and associated with a loss of gut barrier
function [29]. Disruption of these signalling pathways
ameliorates intestinal mucositis. Treatment with NFkB inhibitors has been shown
to partially prevent mucosal injury in an animal model of anticancer treatment [30]. Another anti-inflammatory
agent under development, RDP58, significantly inhibited intestinal damage
induced by irinotecan through its ability to prevent treatment-related
increases in TNF, Interferon-γ, and interleukin-12 [31].  Research is ongoing to fully elucidate the
mechanisms of gastrointestinal mucositis. Loss of crypt stem cells remains an
important event in intestinal injury following anticancer treatment. However, it is not the sole contributing
factor leading to overt damage. The
inflammatory cascade is being realised as an important pathway in the
development of intestinal mucositis that can be pharmacologically manipulated.


## 5. Overview of Current Understanding of Risk Factors for Mucosal Toxicity

Mucosal susceptibility is based on global and tissue-specific
factors. Global components are
associated with treatment and patient characteristics, while tissue specific
components are related to epithelial type, the intrinsic endocrine system, and
the local microbial environment [32]. The choice of drug, the schedule, and
dose-intensity of the regimen will impact on the risk of toxicity [33]. Patient related variables include gender,
ethnicity, and presence of comorbidities such as diabetes mellitus [32], although
the absolute association is far less clear for these variables compared to
treatment. Cellular and molecular elements that influence toxicity will depend
on the local tissue environment, in which mucosal responses to damaging stimuli, whether direct or
indirect through intermediate mediators, are contingent on the particular
location of the tissue and epithelial type, microbial environment and specific
regional functions [32].While the
interactions between global and tissue specific factors impact on mucosal
injury, there
are also underlying genetic influences that profoundly affect toxicity.  The genetic component for predisposition to
mucositis is well established [34, 35].  A classic proof of principle example is the finding
from clinical trials that genetic susceptibility to apoptosis can impact on the
risk of mucositis. Patients with
Addison’s disease, a condition characterised by excess apoptosis, are 17% more
likely to develop oral mucositis during anticancer treatment, and patients with
Psoriasis, a condition of reduced apoptosis especially in skin, are 77% less
likely to suffer oral mucositis [15, 16].Although this is an elegant example, the most widely
accepted evidence for the genetic basis of mucositis risk is the observation
that patients deficient in drug-metabolising enzymes are at a higher risk of
treatment toxicity [32]. Specific examples of these
include deficiencies in UDP-glucuronosyltransferase 1A1 (UGLT), methylenetetrahydrofolate
reductase (MTHFR), thymidylate synthase (TS), and dihydropyrimidine
dehydrogenase (DYDP), during irinotecan, methotrexate, and 5-FU treatment,
respectively [34, 36].
Reduced levels of these proteins can be
caused by hereditable inactivating mutations in the gene promoter region and
result in accumulation and drug prolonged exposure. Pharmacogenetics seems an attractive solution
to explain all mucotoxicity, however the proportion of patients with these enzyme
deficiencies is greatly less than the number of patients that suffer toxicity [2, 32]. It has been suggested that genetic variants
of mucositis mediators, rather than metabolism enzymes, may be associated with
the majority of toxicity.  One such
mediator is TNF. Recent studies have shown that TNF gene
polymorphisms are associated with altered risk of toxicity following cancer
treatment [37, 38]. Patients that are heterozygous for the
TNF-308 promoter polymorphism have increased TNF production and are at a
significantly greater risk of toxicities following myloablative chemotherapy
treatment. Patients had a 17-fold
increased risk of complications following treatment including those affecting the
oral mucosa, skin, and gut, which is a stronger association than traditional
mucositis risk variables of gender, age, and treatment dose [37].  Although
these initial findings are encouraging, the complexity of mucositis means that there
is a continuing challenge to discover more polymorphisms within candidate genes
predictive of regimen-related mucosal damage. These
types of studies provide accumulating evidence to suggest that a number of
genetically controlled elements can determine the response of the mucosa to cancer
treatment, and that these can be either generic or tissue specific (Table 1).Table 1
Genetically controlled elements that may directly or indirectly influence alimentary mucositis risk.
GenericTissue specificDrug metabolism, targets and transportTrefoilsTranscription factorsAdhesion factorsProinflammatory cytokinesDefensinsMediatorsSecretinsSusceptibility to
apoptosis and rateDifferential response to
CT/RTFinally, the
most recent area of research involved in defining risk factors for mucositis is
toxicity clustering. Symptom clustering has previously been applied to a range
of nonmalignant diseases, and to cancer symptoms, and has proven useful for
creating diagnostic criteria. However,
in the context of anticancer treatment-related toxicity, it is in its infancy [39]. The general principle is that a patient who
has alimentary mucositis is likely to have a particular subset of other
toxicities and vice versa. A recent
paper used Bayesian analysis to identify linked toxicities [39] and Markov
networks to define clusters of toxicities [40] in
colorectal cancer patients following treatment.
It found that gastrointestinal toxicities clustered together strongly,  implying that each is a risk factor for the
other and may represent a common underlying aetiology [39]. This novel work has provided the opportunity
to gain a new insight into the relationship of multiple regimen-related
toxicities. Overall, it seems that defining mucosal injury risk factors is as
complex as the pathobiology. The major
components include global, tissue specific, genetic and clustering factors, and
these are combined with cellular and molecular interactions between factors
which ultimately determine the mucosal response to chemotherapy treatment
(Figure 1).Figure 1
Relationship of proposed contributors implicated
in development of alimentary mucositis.


## 6. New Alimentary Mucositis Pathway Research

The
biological events which induce alimentary mucositis begin almost immediately
following administration of radiotherapy and chemotherapy [21, 41]. Inhibition of these early molecular events
may have a profound impact on the intensity of mucosal damage and as such
represents an attractive focus for research.
Applying genomic profiling to mucositis research to investigate global
molecular changes following treatment is an increasing area of interest that
holds much promise. The ability to
simultaneously investigate thousands of genes and their response to treatment
has enhanced our ability to define the mechanisms of damage specific to each
drug and the generic response of tissue to anticancer treatment. We can now
hypothesis
that many genes are initially affected by anticancer treatment which may be
either unique to each drug or common to all drugs. However, these early genes must activate a
smaller subset of downstream genes, involved in specific signalling pathways
that are vital to propagating the cascade towards clinically significant
mucosal damage. What is more, it is
likely that a threshold of key gene activation must be reached before damage
becomes inevitable.Two recent
studies have directed considerable effort towards investigating the gene
expression changes and pathway activation responsible for alimentary mucositis
development [2, 3]. Firstly, Sonis etal. investigated the relationship between gene expression,
canonical pathways, and functional networks in peripheral blood monocytes of
patients who developed mucosal injury in response to chemoradiation [2]. This study combined Bayesian theory with
network analysis to define the canonical pathways most relevant to
regimen-related toxicity. They prioritised
14 pathways derived from the Ingenuity Pathways Analysis Library (Table 2). The study by Bowen etal. investigated gene expression and pathway modulation in the
gastrointestinal tract of rats following irinotecan treatment [3]. They analysed their gene list using web tool,
Pathway Miner, which searches genes based on their associations with cellular
and regulatory pathways and performs a statistical test to rank the most
significant pathways [42]. They found over 20 pathways from the KEGG
database involved in tissue injury following cytotoxic treatment (Table 2). In both studies, Fisher’s exact test was applied to the data
set to determine the most significant pathways associated with toxicity.  Metabolic pathways were also highly
represented in pathway lists and included nitrogen metabolism, oxidative
phosphorylation, purine metabolism, prostaglandin and leukotriene metabolism,
and glutathione metabolism but were not investigated further. Gene association networks are an informative
method for analysing pathway relationships among genes that are coregulated and
for analysing up or down-regulated pathways that contain many participating
genes [42].Table 2
The top 14 cellular and
regulatory pathways deemed to be most relevant to mucosal injury from
anticancer therapy.
RankChemoradiation (Sonis, etal.)Irinotecan (Bowen, etal.)1Toll-like receptor signallingMAPK signalling2NF-kB signallingCell cycle3B-cell receptor signallingComplement and coagulation cascades4PI3K/AKT signallingGap junction5Cell cycleCalcium signalling6P38 MAPK signallingApoptosis7Wnt/B-catenin signallingLeukocyte transendothelium migration8Glutamate receptor signallingVEGF signalling9Integrin signallingCytokine-cytokine receptor interaction10VEGF signallingNeuroactive ligand-receptor interaction11IL-6 signallingWnt signalling12Death receptor signallingB cell receptor signalling13SAPK/JNK signallingT cell receptor signalling14T-cell receptor signallingFocal adhesionThese
papers are the first to use a bioinformatics approach to define the pathways
altered by anticancer treatment during alimentary mucositis development. Despite the use of different databases in the
two studies, there is considerable overlap within the prioritised pathways
named. The difference between sampling
methods (peripheral blood verses gastrointestinal whole tissue) makes the
results even more interesting, as it shows that local tissue damage is also
represented systemically. This has
important implications for future research.
It is also vital that future studies direct efforts towards determining
which signalling pathways are true drivers of damage and which are passengers,
altered without causing a functional change at the tissue level. What is more, the kinetics of pathway
activation needs to
be determined to elucidate which are altered early as damage initiators, which
are upregulated as a consequence of damage, and which of those are crucial to
healing. Each represents a separate
target for intervention.


## 7. Fitting Treatments with Pathways

There are a growing number of
new antimucotoxic agents currently being clinically tested. Mostly these agents target at least one or a
few pathways identified as associated with mucosal injury. However, recent research indicates that only
the agents that modulate multiple pathways will be truly effective at
inhibiting damage. A recent Cochrane Library
review of interventions for preventing oral mucositis found that of the 33
interventions studied twelve showed some evidence of a benefit. Of these, two pharmaceutical agents appear
particularly promising, namely, Amifostine and Benzydamine [43]. These agents have also been included in the
Updated Clinical Practice Guidelines for the Prevention and Treatment of Mucositis
[44]. Amifostine is a free radical scavenger which
exerts its effects by reducing direct DNA damage and reducing upregulation of
inflammatory pathways [45]. It has recently been recommended for use in prevention
of radiation-induced proctitis, however it has yet to be recommended for
mucositis [44, 46]. Benzydamine is recommended for the prevention
of mucositis in radiation patients. It
has anti-inflammatory, analgesic, anaesthetic and antimicrobial effects,
although its primary mode of action is thought to be through inhibition of
proinflammatory cytokines [14]. Mucositis is essentially an inflammatory condition
[47, 48], and the
results of the microarray studies confirm this paradigm by identifying multiple
signalling pathways involved in inflammation.
These include NFkB signalling, complement and coagulation cascades,
toll-like receptor signalling, MAPK signalling, cytokine-cytokine receptor
interaction, as well as others shown in the table above. It seems that these two agents have the
potential to significantly ameliorate mucosal injury through modulating a large
number of regulatory pathways.  Still, Palifermin
(recombinant human KGF-1) remains the only agent currently approved by the FDA for
the prevention and treatment of mucositis [49]. It is biologically pleiotropic. Its primary
mode of action was initially thought to rely on accelerating healing by its
role as an epithelial mitogen enhancing proliferation, migration, and
differentiation of mucosal epithelium. However,
Palifermin has also been shown to exhibit protective functions outside of its
general expected role, including inhibition of epithelial cell apoptosis and
DNA damage, upregulation of detoxifying enzymes, and downregulation of
proinflammatory cytokines [50]. It is likely that Palifermin does this through some of the
pathways discussed. In addition to these drugs, pathway knowledge could be
broadly applied to the development and discovery of new agents effective in
preventing mucosal injury.


## 8. Future Directions for Research

One of the
major challenges in defining the most appropriate molecular targets for
mucositis intervention is the highly complex and interactive nature of its
pathobiology. It seems likely that the
main approach to elucidating the polygenic determinants of treatment will be
genomics, in particular applying the use of anonymous single nucleotide
polymorphism (SNP) maps to perform a genome-wide search for SNPs associated
with treatment effects [51, 52]. This is
opposed to the more traditional technique of singly investigating candidate
genes based on existing knowledge of a drug’s mechanism of action and the known
pathways of metabolism and deposition [51, 52]. This “omic” direction, coupled with powerful
bioinformatics, should greatly advance our ability to unravel the complex
network of mucosal response to treatment in the near future.Since it is
assumed that the body has only a limited number of possible responses to injury
[46], lessons
can also be learnt from other inflammatory conditions. Recurrent aphthous stomatitis (RAS) is an
inflammatory disease of the oral mucosa that is characterised by an aberrant
cell-mediated immune response following external stimuli. The main feature is ulcer formation
precipitated by clinical or subclinical trauma [53]. The
specific immune defect has yet to be identified and the pathogenesis is
unknown. However, a study showed that
there are elevated resting levels of proinflammatory cytokines, particularly
TNF, within nonlesional mucosa of patients with RAS compared to normal controls
and lower levels of the anti-inflammatory cytokine, interleukin-10, in RAS
patients following trauma. Furthermore,
RAS patients all develop ulcers at mucosal trauma sites, while normal patients
show minimal inflammation at the same site [53]. The
proposed mechanisms for this include enhanced T-cell activation together with
an increased sensitivity of keratinocytes to locally derived cytokines. It was proposed to be driven by TNF, while an
absence of anti-inflammatory interleukin 10 (IL-10) may mediate the enhanced
and prolonged injury response [53]. More
recently, a study investigating functional gene polymorphisms in people with RAS showed a marked increase in the IL-1β and TNF heterozygous
genotypes in ulcer sufferers [54]. This indicates a genetic
predisposition to ulcer formation following mucosal injury, and due to the
overlapping features with regimen-related mucosal injury, the implications are that polymorphisms in
inflammatory cascades will also be useful in predicting mucositis risk.Finally, a
critical point that should be addressed before any pathway-targeted treatment can
be developed is the need to define differences in modulation of pathways in
normal tissue that result in toxicity versus the desired effect on tumour
tissue. We must be careful not to interrupt the intended purpose of chemotherapy,
and therefore consideration needs to be given to designing drugs that are
either specifically targeted to the mucosal surface or that exploit features of
the normal cell for drug uptake.


## 10. Conclusions

Alimentary mucositis research has entered the “omic” era. With this has come a new depth of
understanding of the molecular and cellular interactions associated with
development of mucosal injury in response to cancer treatment. One of the most exciting outcomes of recent
research has been the characterisation and prioritising of pathways implicated
in mucositis pathobiology. In the future,
this piece of information will help to rationally design improved drugs and
provide early identification of patients at risk of developing severe
mucositis.



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