Introduction
Head and neck cancers (HNCs) are the fifth most common
cancers in the world. More than 50% of the cases
are diagnosed during the locally advanced disease
stage. Chemoradiotherapy (CRT), which is administered
as a primary or postoperative adjuvant approach
in the treatment of HNC, has become a standard therapy
since 10 years due to its successful results in providing
a locoregional control and preventing metastasis.
For the therapy, one or more chemotherapeutic agents
may be used concomitantly with radiotherapy (RT).
The most commonly used agents are cisplatin, 5-fluorouracil
(5-FU), mitomycin C, and taxane.[,,] Oral
mucositis is the most important side effect occurring
during the treatment, which leads to an interruption
in the treatment of patients by impairing the oral intake
and causing malnutrition. The interruption of the
therapy due to oral mucositis results in prolongation
of the total duration of the therapy, which eventually
leads to tumor repopulation and a decrease in the rate
of locoregional control.[] Due to RT, a local inflammation
and an acute-phase response occur because of
cell damage in the irradiated volume. This condition
is caused by an increase in the levels of proinflammatory
cytokines [tumor necrosis factor (TNF)-alpha and
interleukin (IL)-6]. Local inflammation and irritation
form the basis for the development of oral mucositis
in patients with HNC.[] Glutamine is a nonessential
free amino acid that can be synthesized in the body. It
is the precursor of nucleotides in DNA and RNA synthesis
and plays a key role in lymphocyte proliferation.
Glutathione (GSH/glutamyl-cysteinyl-glycine), which is
the byproduct of GLN metabolism, is one of the antioxidants
that have the highest concentrations in the cell and
protects the normal tissue against oxidative damage.[]
The level of GSH in tumor cells is 5-50 times higher
than that in nontumor cells, and this high level of GSH
mediates the resistance to CT and RT.[,,,] The resistance
for many anticancer agents is generally associated
with increased levels of GSH in the tumor [,];
therefore, it is thought that reducing the levels of GSH
in the tumor will increase the sensitivity of the tumor
for RT and CT. In many studies, it has been shown that
oral intake of GLN paradoxically reduces the levels of
GSH in the tumor and increases them in the normal
tissue.[,,,] This is very important because
GSH, which is the key regulator of cellular redox, determines
the response of the tumor to CT and RT (simultaneously
protects the normal cells).[,,] GLN
supplementation reduces the levels of intratumoral GSH and thereby prevents the continuation of tumor
response to cytotoxic therapy.[,,,] This information
suggests that GLN protects the normal tissue
by increasing the selectivity of the antitumor therapy
for the tumor and results in chemotherapeutical damage
in the tumor cells by sensitizing them. As a result,
a clear increase is seen in the therapeutic index. Few
randomized studies have investigated the role of glutamine
supplementation in the prevention of oral mucositis
due to RT or CRT. These studies revealed that
the prophylactic intravenous or oral use of glutamine
decreased the intensity and duration of oral mucositis.
[,] In cell cultures, glutamine affected the production
of cytokines such as TNF-alpha and IL-6, which are released
from macrophages and neutrophils.[,]
This study aimed to investigate the effect of glutamine
supplementation on the intensity and duration of
oral mucositis related to therapy and on proinflammatory
cytokines in patients with HNC who were treated
with CRT.
Methods
A total of 28 outpatients with HNC (24 male and 4 female)
who were admitted to the Gazi University Department
of Radiation Oncology between October 2008
and November 2009 were retrospectively evaluated. The
mean age of the patients was 18-76 years. Of the patients
with an Eastern Cooperative Oncology Group (ECOG)
performance score of 0?3 at presentation to the clinic,
12 had nasopharyngeal, 11 had laryngeal, five had oropharyngeal,
and one had hypopharyngeal carcinoma. At
the time of diagnosis, all the patients had an ECOG performance
score of 0-2, except one patient with a locally
advanced hypopharyngeal carcinoma who had a score
of 3. All the patients were histopathologically diagnosed
with squamous cell carcinoma.
Of the 28 patients who were included in the RT program,
five were postoperatively included and 23 were
included according to organ protection protocols. While
two patients received RT alone postoperatively, 26 (92%)
received chemotherapy (CT) regimens concomitantly
with RT, and 6 (20%) had received induction CT before
concomitant CT. Of the 26 patients who received CT, 13
received cisplatin, 11 received cisplatin and docetaxel,
one received cisplatin, docetaxel, and 5-FU, and one received
carboplatin concomitantly with RT.
2.1. Radiotherapy
After the immobilization of all patients using a thermoplastic
fixation mask, their shoulders were pulled out using a string to exclude them from the therapy. After
positioning the patient, treatment planning images were
obtained using computed tomography (CT). The data
obtained were transferred to the radiotherapy therapy
planning system called Eclipse 7.2.02 (Varian Medical
Systems, Canada, USA) in our clinic and were combined
to obtain a 3D image. All targeted volumes, such
as gross tumor volume, clinical targeted volume (CTV),
and planned targeted volume, were drawn by a radiation
oncologist according to ICRU-62 recommendations using
axial CT images. TV was defined by considering the
tolerated doses for critical organs, such as the spinal cord
and mandible. RT was administered at doses and to sites
which are routinely used in the clinical practice: conventional
doses of 2 GY daily for every week and as five fractions
per week. While one patient with a postoperative
RT indication received 50 Gy to complete the therapy,
four had an increase of the dose to 60-64 Gy at the primary
tumor site by boosting after the administration of
50 Gy. All the patients who were included in the organ
protection protocol received a total of 70 Gy at the primary
site with a boost after the administration of 50 Gy.
2.2 Administration of glutamine
Eighteen patients prophylactically received oral powdered
L-glutamine (Glutamine ResourceR, Nestle)
starting from the first day of RT at a total dose of 30
g/day (10 g at each 8 h, by mixing with water or fruit
juice). For these patients, glutamine supplementation
was continued until the completion of RT. Ten patients
did not receive any supplementation product.
2.3. Evaluation of patients and oral mucositis scoring
At the initiation of RT, height and weight were measured
for all the patients. The grade of oral mucositis
was evaluated using the Radiation Therapy Oncology
Group (RTOG) scoring system. During RT, the changes
in the weight of the patients were routinely evaluated,
and oral mucositis scoring was performed every week.
At the beginning, during, and at the end of the therapy,
serum IL-1 beta, IL-6, and TNF-alpha levels were measured,
in addition to performing routine blood tests.
2.4. Statistical analysis
For statistical analysis, patients were stratified according
to their demographic characteristics and use of glutamine
(yes/no). For the stratification, parameters such as
age, sex, ECOG performance status, induction CT, and
stage of the disease were used. While the distribution of
frequency was used to describe the categorical variables,
mean, median, and range were used for quantitative
variables. Frequency distributions were compared using
the Mann-Whitney, Chi-square, and Student's t test. A
p-value of <0.05 was considered significant.
Results
In this retrospective study, 28 eligible patients were
identified, and all the patients were included in the
statistical analysis. None of the patients showed glutamine
intolerance or glutamine-related toxicity. Table
1 shows the comparison between the therapy groups
determined according to the use of glutamine (yes/no), by the parameters formed before the therapy. No statistically
significant differences were found between the
two groups. Table 2 shows the results of the therapy.
Table 1: Pretreatment patient and tumor characteristics
Table 2: Therapeutic results
3.1. Oral mucositis
The incidences of grade 2 mucositis were comparable
between the groups who received and did not receive
glutamine supplementation (42.1% and 44.4%, respectively).
However, the incidence of severe mucositis
(RTOG of ?grade 3) was significantly lower in the
group receiving glutamine supplementation than in the
group not receiving it (5.3% and 55.6%, respectively;
p=0.008). In the group which did not receive glutamine
supplementation, two patients were hospitalized due to
severe mucositis. In the group which received oral glutamine,
grade 1 toxicity was most commonly observed,
with a rate of 47.4%. The day of onset of mucositis was
the 18th day of the therapy in the group that received
glutamine supplementation and 14th in the group that
did not receive glutamine supplementation; this was
statistically significant (p=0.007). Similarly, the median
dose at which mucositis was started was 36 Gy in the
group that received glutamine supplementation and 28
Gy in the group that did not receive glutamine supplementation
(p=0.006).
3.2. Weight loss and BMI change
The two therapy groups did not show any statistically
significant difference in terms of weight change (kg),
percentage of weight change, and BMI (kg/m2) when
the changes in weight were examined at the beginning and end of the treatment (p=0.42, p=0.47, and p=0.44,
respectively).
3.3. Cytokine levels
When the changes in serum TNF-alpha, IL-1 beta, and
IL-6 levels were compared at the beginning and end
of the treatment, the groups that received and did not
receive glutamine supplementation did not show any
significant difference (p=0.16, p=0.08, and p=0.77, respectively).
Discussion
In patients with HNC who were treated with CRT, glutamine
supplementation delayed the clinical onset of
oral mucositis due to the therapy and reduced the intensity
of mucositis. However, when weight changes of
all the patients were evaluated at the beginning and end
of the therapy, the two groups did not show any significant
difference in terms of weight loss.
In patients with HNC, the therapy includes RT,
surgery, and CT, if required. Local inflammation and
irritation related to RT results in side effects, such as
mucositis, xerostomia, odinophagia, stomatitis, oesophagitis,
and dysphagia.[,] Oral mucositis is the
most important side effect in which the clinical symptoms
are induced as a result of a loss in the epithelial
cells. In patients who receive RT, mucositis generally
appears within the second week of the therapy and
continues until the end of the therapy with a gradually
increasing intensity. In patients who receive CT, it appears within 3-5 days after initiation of the therapy
and peaks on days 7-10. Currently, there are no established
methods to prevent or treat oral mucositis.
For its treatment, many agents such as sucralphate,
amiphostine, chlorhexidine, antimicrobial agents, and
granulocyte-macrophage colony stimulating factor are
being used, but the benefits of these agents in preventing
or treating oral mucositis due to CRT are limited.
[,,]
Glutamine is a free amino acid that can be synthesized
in the body. It is the precursor of both glutamate,
which is required for the synthesis of glutathione and
protects the cells from oxidative stress, and nucleotides
playing a role in RNA and DNA synthesis. In
their study, Klimberg et al. demonstrated the efficacy
of glutamine, which is the main energy source of the
intestinal epithelium, in the prevention of mucosal
damage caused by radiation exposure in the intestine
of rat models.[,,] In addition, in the rats treated
with methotrexate, the group that received glutamine
supplementation showed a decrease in weight loss, increase
in the mucosal weight of the jejunum and colon,
longer lifespan, and lower incidence of bacteremia.[]
Many clinical studies have investigated the efficacy of
glutamine in patients diagnosed with cancer during the
last 20 years. Anderson et al. in their nonrandomized
study which included 24 patients who received intensive
CT regimens before bone marrow transplantation
demonstrated that prophylactic use of oral glutamine
reduces the intensity and duration of oral mucositis
due to CT.[] Recently, Peterson et al. in a randomized,
placebo-controlled, multicenter, phase 3 study
conducted on 326 patients with breast cancer receiving
CT demonstrated that the clinical incidence and intensity
of mucositis were significantly reduced in patients
using glutamine compared with the placebo.[]
When the Western style of nutrition is taken into
consideration, dietary daily glutamine intake is generally
<10 g. In humans, some catabolic conditions such
as trauma and sepsis increase the need for glutamine,
and quantity of daily dietary glutamine intake required
to maintain homeostasis increases up to 20-40 g.[] In
patients with HNC, glutamine deficiency is an expected
result. This leads to more severe manifestations of
side effects that occur during the therapy. Because patients
diagnosed with HNC have a history of smoking,
use of alcohol, and malnutrition in general, deficiency
of glutamine in addition to all these factors reduces
the tolerance of the normal tissues to antineoplastic
therapies.[,] The first study investigating the effect
of glutamine on oral mucositis in patients receiving RT or CRT for HNC was conducted by Huang et al.,
wherein 17 patients who received RT alone for HNC
were randomized according to their use of prophylactic
oral glutamine (yes/no). In this study conducted
with a limited number of patients, it was found that the
duration and intensity of oral mucositis significantly
decreased in the group that received oral glutamine
compared with the placebo group (p≤0.006).[] In
the double blind, placebo-controlled study of Cerchietti
et al. that included 32 patients who received CRT
for HNC, patients were given L-alanine?L-glutamine
combination via the intravenous route; the study found
that the intensity of mucositis was significantly lower
in the group receiving L-alanine?L-glutamine than
in the placebo group (p=0.007).[] In this retrospective
study, 28 patients who received primary or adjuvant
CRT due to the diagnosis of HNC were evaluated.
The patients who received oral glutamine during the
therapy were compared with those who did not receive
any nutritional supplementation. The patients who received
glutamine supplementation were administered
this product at a dose of 30 g/day as a prophylaxis starting
from the beginning of the study. Parameters such
as time of onset of mucositis due to the therapy, dose
of radiotherapy at the time of first diagnosis, and severity
of mucositis were investigated. The evaluation
showed that glutamine supplementation in patients
receiving CRT for HNC significantly delayed the onset
and decreased the intensity of mucositis. In the group
that did not receive glutamine supplementation, two
patients were hospitalized due to severe mucositis after
the interruption of their therapy. However, there were
no patients who interrupted the therapy due to severe
mucositis or other reason in the group who received
the supplementation. The incidence of severe mucositis
(≥grade 3) was approximately 5% in the group that
received the supplementation and approximately 55%
in the group that did not receive the supplementation
(p=0.008). The two groups did not show any statistically
significant difference in terms of weight loss due to malnutrition.
This result was similar to the results obtained
from the study of Huang et al. Thus, glutamine alone
cannot prevent weight loss which gradually increases
during the therapy. Therefore, an additional nutritional
supplementation along with glutamine in patients with
HNC may be efficient for the prevention of weight loss.
It is known that glutamine plays a key role in lymphocyte
proliferation and affects the production of cytokines
such as TNF-alpha and IL, which are released
from macrophage and neutrophils. Pithon-Curi et al.
demonstrated the effect of glutamine on cytokines levels in cell cultures.[,] Recently, Cruzat et al.
found that plasma concentrations of TNF-alpha were
lower in the rat groups who received free glutamine
and L-alanine-L-glutamine dipeptide supplementation
for the inflammatory response due to long term
exercise than those in the control group (p<0.05).[]
Similarly, Fan et al. found that TNF-alpha were lower
in burned rats who were given glutamine than in the
controls (p<0.05).[] In their animal study, Ameho
et al. investigated the effect of prophylactic glutamine
on the occurrence of colitis, which develops in rats after
exposure to trinitrobenzene sulphonic acid. In this
study, although there was less damage and less bacterial
translocation in the group which received GLN
prophylaxis, the levels of IL-8 and TNF-alpha, which
are strong inflammatory cytokines, was significantly
lower than those in the control group.[]
In HNC, locally administered RT leads to an inflammatory
response in the irradiated volume. Due
to cell damage, a local inflammation and an acutephase
response are obserevd.[,] These conditions
are caused by increased levels of proinflammatory
cytokines (TNF-alpha and IL-6).[,] In our study,
in all the patients, serum TNF-alpha, IL-1b, and IL-6
levels were measured at the beginning and end of the
therapy, and responses for the following questions were
elucidated: "In patients who receive RT for HNC, does
glutamine decrease the inflammation due to RT? Is the
positive effect of glutamine on oral mucositis correlated
with the levels of these cytokines?" The serum levels of
IL-6 measured at the end of the therapy were increased
compared with those at the beginning of the therapy
in both the groups. The levels of TNF-alpha and IL-1b
at the end of the therapy were clinically reduced in the
group that received glutamine supplementation compared
with the group that did not; however, this was
not statistically significant. This statistical result may
be due to the limited number of patients evaluated and
an unequal distribution of patients between the groups.
Based on this retrospective evaluation, we believe
that there is a need for prospective, randomized studies
including large patient samples to investigate the
association between increased serum proinflammatory
cytokine levels due to RT and prophylactic glutamine
supplementation for reducing the intensity of mucositis
in patients with HNC.
Conclusion
Glutamine is a well-tolerated supplementation product
with proven effects on oral mucositis, which is the side effect occurring during the treatment of HNCs. The effect
of glutamine on inflammation leading to mucositis
development in patients with HNC should be investigated
in larger patient populations.
Peer-review: Externally peer-reviewed.
Conflict of Interest: None declared.
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