Lasers in Surgery and Medicine 47:296305 (2015)

Effect of Low-Level Laser Therapy on

Chemoradiotherapy-Induced Oral Mucositis and Salivary
Inflammatory Mediators in Head and Neck Cancer Patients

lica F. Oton-Leite, DDS, MS, PhD,1 Geisa Badauy L. Silva, DDS, MS, PhD,1 Mar
lia O. Morais, DDS, MS,1
Ange

udio R. Leles, DDS, MS, PhD,3 Marize Campos Valadares, BS, MS, PhD,4
Tarc
lia A. Silva, DDS, MS, PhD,2 Cla
5
Juliana Castro D. Pinezi, MD, MS, Aline C. Batista, DDS, MS, PhD,1 and Elismauro F. MendonSc a, DDS, MS, PhD1

1
Department of Oral Medicine (Oral Pathology), Dental School, Federal University of Goi

as, Goi^
ania, Goi

as 74605-220,
Brazil
2
Department of Oral Medicine (Oral Pathology), Dental School, Federal University of Minas Gerais, Belo Horizonte,
Minas Gerais 31270-901, Brazil
3
Department of Prevention and Oral Rehabilitation, Dental School, Federal University of Goi

as, Goi^
ania,
Goi

as 74605-220, Brazil
4
Laboratory of Cellular Pharmacology and Toxicology, Faculty of Pharmacy, Federal University of Goi

as, Goi^
ania,
Goi

as 74605-220, Brazil
5

Jorge Hospital, Association of Cancer Combat of Goi

Radiotherapy Department of the Araujo
as, Goi^
ania,
Goi

as 74605-070, Brazil
Background and Objective: Oral mucositis (OM) is
considered a painful and debilitating side effect in patients
receiving head and neck cancer treatment. Low-level laser
therapy (LLLT) proved to be effective to prevent and treat
chemoradiotherapy-induced OM. The aim of this study was to
evaluate the effect of LLLT in the severity of OM in patients
with head and neck cancer and on the release of salivary
inflammatory mediators. Clinical (score of OM severity) and
biochemical parameters (concentration of inflammatory
mediators, growth factors, and enzymes in saliva) were used.
Materials and Methods: Thirty patients were randomized into two groups: control and laser. LLLT was
performed three times a week in the laser group, while
control group received sham irradiation. OM severity was
assessed according to the World Health Organization
(WHO) and National Cancer Institute (NCI) scales. Proinflammatory and anti-inflammatory cytokines (TNF-a,
IL-6, IL-1b, IL-10, TGF-b), growth factors (EGF, FGF,
VEGF), and metalloproteinases (MMP2/TIMP2, MMP9/
TIMP2) concentrations were assessed using ELISA test.
Saliva samples were collected on admission, and at the 7th,
21st, and 35th sessions of radiotherapy.
Results: The laser group showed a reduction in the
severity of OM, which coursed with significantly diminished salivary concentration of EGF and VEGF in the 7th
radiotherapy session and of IL-6 and FGF in the 35th.
There was a trend for reduced levels of IL-1b, TNF-a, IL10, TGF-b, MMP2/TIMP2, MMP9/TIMP2 in the laser
group compared to the control, however, no statistically
significant differences were found.
Conclusions: These findings demonstrated that LLLT
was effective in reducing the severity of chemoradiotherapy-induced OM and was associated with the reduction of
inflammation and repair. Lasers Surg. Med. 47:296305,
2015. 2015 Wiley Periodicals, Inc.
2015 Wiley Periodicals, Inc.

Key words: cytokines; growth factors; head and neck

cancer; laser therapy; matrix metalloproteinases
INTRODUCTION
Oral mucositis (OM) is an inflammatory event, characterized by painful and debilitating lesions, it is considered as side
effects of the chemotherapy and radiotherapy (RT) used in the
treatment of patients with head and neck cancer [1,2]. It is a
dose-limiting toxicity associated with pain, nutritional compromise, and potential risk of secondary infection, which can
lead to unplanned treatment interruption, compromising the
likelihood of cure and the quality of life of patients [3,4].
There is a consensus that an interruption in RT, even if
only for a few days, is sufficient to contribute to the
differentiation process and to the proliferation of the
remaining tumor cells and thereby promotes tumor
growth [3,5]. Furthermore, treatment interruption increases the chances of regional recurrences which reduce
the chances of cure and survival of patients with head and

Conflict of Interest Disclosures: All authors have completed

and submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest and none were reported.
Contract grant sponsor: National Council for Scientific and
Technological Development; Contract grant number: 479587/
2010-8; Contract grant sponsor: Foundation for Research Support in the State of Goi

as; Contract grant number:
2010102670007.


Correspondence to: Dr. Elismauro F. Mendonca, DDS, MS,
PhD, Faculdade de Odontologia da Universidade Federal de
Goi

as, Praca Universit

aria, S/N, Setor Universit

ario, Goi^
ania,
Goi

as 74605-220, Brazil. E-mail: elismaur@ufg.br
Accepted 1 February 2015
Published online 30 March 2015 in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/lsm.22349

EFFECT OF LOW-LEVEL LASER THERAPY IN CANCER PATIENTS

neck cancer [5]. Therefore, there is a need to control

the adverse effects of adjunctive therapies in the oral
cavity [3,5].
Studies have confirmed the complexity of mucositis
pathogenesis and its association with microvascular
injury [6], pro-inflammatory cytokines [7,8], hostmicrobiome interactions [9], and extracellular matrix alterations [10]. It is now clear that mucosal injury results from
a series of inflammatory events in which cellular mediators
play a crucial role [2]. Of these, the tumor necrosis factor
alpha (TNF-a), interleukin-1beta (IL-1b), and interleukin6 (IL-6), all pro-inflammatory cytokines, are involved in
oral and gastrointestinal mucositis [2].
Other mediators involved in the ulcerative phase of OM and
the healing process are matrix metalloproteinases (MMPs)
and growth factors, such as the epidermal growth factor
(EGF), the fibroblastic growth factor (FGF), and the vascular
endothelial growth factor (VEGF) [10,11]. Studies have shown
changes in the extracellular matrix attributed to the
deregulated expression of MMPs following cancer treatment [10,11]. In contrast, growth factors help to maintain
homeostasis and play an important role in wound healing [11].
In most patients the management of OM is centered on
relief of symptoms. A large number of agents have been
indicated to deal with OM, for instance, topical antimicrobial agents, vitamins, growth factors, mouth washes, but
generally with inconsistent results [12,13].
Nowadays, Low Level Laser Therapy (LLLT) is considered a promising therapy. It has been used in the
prevention and treatment of OM in several clinical
settings, including RT for head and neck cancer patients
and in patients receiving high-dose chemotherapy in cases
of hematopoietic stem cell transplantation [1422]. The
Multinational Association of Supportive Care in Cancer/
International Society of Oral Oncology (MASCC/ISOO)
recently completed the process of updating the guidelines
for the prevention and treatment of mucositis and
suggested the use of LLLT to prevent OM in head and
neck cancer patients undergoing RT, but makes no specific
recommendation about the use of laser for chemoradiotherapy-induced mucositis [23]. In general, these studies
have shown that LLLT is safe and has anti-inflammatory,
analgesic, and biomodulatory effects.
Although earlier studies have reported the use of LLLT in
the prevention and treatment of OM, the mechanism of
action of this therapy is still not well understood [1422].
Therefore, based on the pathogenesis of OM, the aim of this
study was to evaluate the effect of LLLT in the severity of
OM and on the release of salivary molecules (TNF-a, IL-1b,
IL-6, IL-10, TGF-b, EGF, FGF, VEGF, MMP2/TIMP2,
and MMP9/TIMP2) during chemoradiation for head and
neck cancer.
MATERIALS AND METHODS
Study Design and Patients Selection
This study was a randomized, double-blinded, placebo
controlled clinical trial. Fifty head and neck cancer
patients scheduled to receive chemoradiotherapy were

297

recruited from April 2011 to December 2012 at a reference

hospital for cancer treatment (Ara

ujo Jorge Hospital,
Goi

as Cancer Combat Association, Goi^
ania, Brazil). No
patient had salivary gland tumors, or had a previous
history of chemotherapy and/or RT in the head and neck
region. The study protocol was approved by the local Ethics
Research Committee (Protocol 36/08) in accordance with
the Helsinki Declaration. All patients were aged 18 years
or over and signed an informed consent form to participate
in the study.
All patients were submitted to conventional RT treatment (two dimensional techniques), covering a wide area of
the head and neck region, in general using a parallelopposed two field technique. The RT protocol of the hospital
for head and neck cancer patients consisted of treatment
sessions 5 days a week, 2 Gy per fraction, with a total dose
of 70 Gy over 7 weeks (35 sessions). A minimum dose of
50 Gy was delivered to the oral cavity of all patients in the
study. In addition, all received concomitant chemotherapy
with cisplatin (100 mg/m2 every 21 days).
Blocked randomization was performed to allocate 30
patients into two groups, one which would be irradiated
with LLLT (laser group) and one which received sham
irradiation (control). The randomization of the study is
shown in Figure 1.
Both groups received standard oral health care for
preventing and minimizing the oral complications of
chemoradiotherapy and antifungal and/or analgesic medication when needed. In addition, patients with acute
general dental needs were treated before starting the
chemoradiotherapy protocol.
Laser Therapy
The laser group was treated with an InGaAlP diode laser
(Twin Flex Evolution, MMOptics Ltda, S~
ao Carlos, Brazil),
operating at 660 nm, 25 mW output power, in a continuous
wave, at a fluence of 6.2 J/cm2, energy per point of 0.24 J.
Irradiation time was 10 seconds in each point based on the
laser spot size of 0.04 cm2. Laser energy was applied
punctually and perpendicular in contact with the tissue.
Low level laser irradiation was carried out in the following
sites of oral cavity: buccal mucosa (ten points on each side),
lips (eight points on upper and lower internal mucosa),
hard palate (three points), lateral tongue (ten points on
each side), dorsal tongue (three points), soft palate (three
points), floor of the mouth (two points), and in the labial
commissure (one point on each side). It is relevant to take
into account that the surgical bed (tumoral area removed)
was not involved in the field of laser irradiation.
In both groups, laser irradiation was performed three
times a week on alternate days over 7 weeks (35 sessions).
All sessions were conducted by the same operator. The
active tip of the laser was disinfected with 70% alcohol and
covered with a plastic film. All patients wore protection
eyeglasses and kept their eyes closed to make sure that
they would not know if they were receiving laser
irradiation or sham-laser (laser light inactived). The
same procedures and equipment were used in both groups,

298

OTON-LEITE ET AL.

Fig. 1. Patient flow diagram.

except for the fact that patients of the control group

received sham-laser.
The first session was performed at the first day of RT,
and the following sessions occurred three times a week on
alternate days, always before each session of RT, until the
end of the treatment. Just to emphasize, all patients
received concomitant chemotherapy during RT.
Oral Mucositis Evaluation
OM was evaluated weekly by the same trained dentist,
blinded to the randomization. The operator who performed
the laser irradiation did not participate in the OM
evaluation.
The severity of OM was rated according to the National
Cancer Institute (NCI) and the World Health Organization
(WHO) grading of mucositis scales. These classifications
are widely used both in routine clinical practice and in
clinical trials involving OM. During the oral examination,
OM severity was rated as follows:
1. NCI scale: Score 0oral mucosa presenting no visible
alteration; Score 1presence of erythema; Score 2

ulcers up to 1.5 cm in diameter and noncontiguous;

Score 3ulcers larger than 1.5 cm in diameter and
contiguous; and Score 4ulcers showing necrosis and
bleeding.
2. WHO scale: Score 0no signs or symptoms; Score 1
oral soreness and erythema; Score 2oral erythema
and ulcers, both solid and liquid diets tolerated; Score
3oral ulcers, liquid diet only; Score 4oral alimentation impossible.

Saliva Collection
Saliva samples were obtained at one point before
chemoradiotherapy and at three points during the
treatment (7th, 21st, and 35th sessions of RT) according
to the spitting method proposed by Navazesh [24]. Patients
could not eat or drink an hour before saliva collection
procedure. They washed the mouth with pure water and
swallowed whole saliva before collection.
For unstimulated saliva collection, patients were instructed to slightly tilt their head down and then they
should expectorate the saliva, which was produced over a

EFFECT OF LOW-LEVEL LASER THERAPY IN CANCER PATIENTS

5 minute period, into a plastic sterile tube. The unstimulated salivary flow was measured in milliliters per minute
(ml/min).
The saliva samples were subsequently diluted (1:1) in a
phosphate-buffered saline (PBS) solution containing protease inhibitors (0.1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 0.1 mmol/L benzethonium chloride, 10 mmol/
L EDTA, 0.01 mg/ml aprotinin A, and 0.05% Tween 20) and
subsequently frozen at 808C to await analysis.
Enzyme Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) was
performed as previously described [24]. Using commercially available quantitative sandwich ELISA kits (DuoSet, R&D Systems, Minneapolis, MN) in accordance with
the manufacturers instructions, the salivary levels of the
inflammatory mediators were measured with the following
components: TNF-a, IL-1b, TGF-b, IL-10, EGF, FGF,
VEGF, MMP2/TIMP2, and MMP9/TIMP2. A High Sensitivity kit was used for IL-6. The samples were analyzed and
the values were determined based on a negative control
(PBS) and a standard curve, which were provided by the
manufacturer. The Bradford assay with a bovine serum
albumin standard (Fermentas Life Sciences, Vilnius,
Lithuania) was used to measure the total protein
concentration of the saliva samples expressed as milligram
per milliliter. This total protein concentration was used to
adjust the saliva cytokine values for each sample. The
saliva sample values, corrected by the total protein, were
expressed in picogram per milligram protein.
Statistical Analysis
The patients demographic and clinical characteristics
were compared across both groups (control and laser) using
the Pearson x2 test (categorical variables; Table 1). The
Mann-Whitney test was used to compare control and laser
groups when data were not normally distributed. Statistical significance was set at P < 0.05. SPSS 17.0 software
(SPSS, Inc., Chicago, IL) and Prism (Graph Pad Prism
5.01) were used for data analysis.
RESULTS
Patient Characteristics
All 25 patients in the study had a diagnosis of primary
squamous cell carcinoma of head and neck. In the laser
group, three patients died during the course of the study,
two of them developed pneumonia, and the other died due
to hemorrhagic complication while in the control group;
two patients dropped out of treatment.
All patients with oral cancer underwent surgical
excision of the tumor before starting the protocol of
chemoradiotherapy. All patients showed a decrease in
salivary flow rates without any significant differences
between the groups in all periods evaluated (P > 0.05).
There were no differences between the groups with regard
to age (P 0.14), gender (P 0.32), or tumor location
(P 0.85). No significant side effects attributed to the laser

299

TABLE 1. The Mann-Whitney Test was Used to Compare Control and Laser Groups When Data were not
Normally Distributed
Group frequency
Patient characteristics
Age
60 yrs
<60 yrs
Gender
Male
Female
Primary tumor
Oral cavity
Pharynx
Oropharynx
Surgery
Yes
No
T-stage
T1/T2
T3/T4
Tx
N-stage
N0/N1
N2/N3
Nx
*

Laser

Control

P-value*

3
9

7
6

0.14

9
3

12
1

0.32

5
3
4

4
4
5

0.85

7
5

5
8

0.32

5
6
1

3
10
0

0.16

6
4
2

4
9
0

0.21

Pearson x2 test (P > 0.05).

therapy were observed. The main characteristics of the

study sample are presented in Table 1.
Effect of LLLT on Occurrence and Severity of OM
According to the NCI classification, the incidence of
grade 2 OM at the 7th RT session was 25% in the laser
group (3 out of 12 patients) and 76.9% in the control group
(10 out of 13 patients). At the 21st RT session, six patients
(50%) showed grade 3 in the laser group and 12 in the
control group (92.3%). In the last clinical evaluation of OM,
25% of patients had grade 3 in the laser group (3 out of 12)
and 53.8% (7 out of 13) in the control group, due to severe
OM in the 21st session.
The results showed that the OM grades in both scales
(WHO and NCI) were significantly lower in the laser group
than in the control group at the 7th, 21st, and 35th RT
sessions (P < 0.05; Fig. 2A and B).
Salivary Inflammatory Mediator Levels
Pro-inflammatory and anti-inflammatory cytokine levels. Salivary normalized cytokine levels were
analyzed in resting saliva for both the laser and control
groups.
In the control group, levels of IL-6 followed the clinical
course of OM until the peak (ulcerative phase) at the 21st
RT session. Interestingly, salivary IL-6 concentration in
the group of patients undergoing LLLT decreased

300

OTON-LEITE ET AL.

difference was not statistically significant (P > 0.05 for all

comparisons; Table 2).
Growth Factors Levels
For the growth factors analyzed, FGF levels decreased
after the ulcerative phase of OM in the laser group. This
reduction was statistically significant at the 35th session,
corresponding to wound healing (P 0.038).
Lower levels of EGF and VEGF were observed in the
laser group at the 7th RT session when compared to the
control group (P 0.04 for EGF and P 0.035 for VEGF).
No significant changes were seen in relation to the other
periods evaluated in either groups. These results are
illustrated in Table 2.
In addition, an interesting finding was the increased
release of growth factors (FGF, EGF, and VEGF) in the
control group after the peak of OM, although no significant
difference was found (P > 0.05).
Matrix Metalloproteinases Levels

Fig. 2. Clinical evaluation of OM severity in head and neck cancer

patients during chemoradiotherapy. Statistically significant
differences between the groups were found when patients
completed 7th, 21st, and 35th sessions of radiotherapy. A:
represents the WHO classification of OM and B: the NCI
classification. (x2 test, *means P < 0.05).

significantly at the end of RT (35th session) after 21

sessions of laser therapy (P 0.032; Fig. 3).
In addition, the laser group tended to have lower
salivary concentrations of the pro-inflammatory cytokines,
IL-1b and TNF-a, in the ulcerative phase of OM (21st
session) and at the end of RT (35th session) when compared
to the control group. However, this difference was not
statistically significant at any of the time-points evaluated
(P > 0.05 for all comparisons; Table 2).
With regard to the anti-inflammatory cytokines IL-10
and TGF-b, the data showed a slight reduction for those
patients undergoing LLLT when compared to the control
group at almost all the times evaluated; however, this

Fig. 3. Salivary IL-6 concentration gradually decreases from the

1st week (7th session) to the 7th week (35th session) of
radiotherapy in the laser group when compared to the control
group. Statistically significant difference was found in the 35th
session (*means P 0.032).

There was a reduction of MMP9/MMP2 levels in the

laser group compared with the control mainly after the
peak of OM, but no statistically significant difference was
found (P > 0.05; Table 2). MMP2/TIMP2 release in the
control group was higher than in the laser group mainly at
the peak of OM, although not statistically significant
(P 0.49). After this period, a decrease in MMP2/TIMP2
levels was found in both groups but without significant
difference (P > 0.05; Table 2).
DISCUSSION
Some clinical trials have investigated the clinical course
and pathophysiology of OM [2533]. Recently, Silva
et al. [25] demonstrated that patients undergoing hematopoietic stem cell transplantation (HSCT) exhibited a peak
of OM 9 days after transplantation (D 9), corresponding
to about 1114 days after the onset of high dose
chemotherapy, depending on the conditioning regimen of
each patient, and wound healing often occurred on marrow
engraftment day, between 11 and 18 days after transplantation. With regard the course of OM in our study,
involving patients receiving concomitant chemotherapy
with RT for head and neck cancer, different results have
been observed, the peak of OM severity was after 21
sessions of RT and patients showed a longer healing time
for ulcers, even with the conclusion (end) of the RT, the
damage effect in tissue remains for days. As we can see,
different clinical course for OM are seen in different
treatment protocols for oncologic patients suggesting
different pathways to OM pathophysiology.
In relation to pathophysiology of OM, some differences
between chemotherapy and chemoradiotherapy-induced
OM may also be highlighted. High levels of growth
factors were observed until 7 days after transplantation
(D 7) in study of Silva et al. [25], whereas in head and
neck cancer patients there was an increase of FGF, EGF,
and VEGF levels after the peak of OM. In addition,
MMP9 release increased gradually until the peak of

EFFECT OF LOW-LEVEL LASER THERAPY IN CANCER PATIENTS

301

TABLE 2. Cytokines, Growth Factors, and MMPs Concentrations in Saliva on Baseline, 7th, 21st, and 35th Days of
Radiotherapy (RT) in Head and Neck Cancer Patients Receiving Laser Therapy (Laser Group) and in the Control
Group
Evaluation periods
Groups
TNF-a
Laser
Control
P-value
IL-1b
Laser
Control
P-value
TGF-b
Laser
Control
P-value
IL-10
Laser
Control
P-value
FGF
Laser
Control
P-value
EGF
Laser
Control
P-value
VEGF
Laser
Control
P-value
MMP2
Laser
Control
P-value
MMP9
Laser
Control
P-value

Baseline

7th RT

21st RT

35th RT

13.7 (065.6)
19.9 (081.2)
0.43

19.3 (069.8)
17.3 (046.1)
0.91

13.5 (042.9)
31.9 (0295.2)
0.69

10.5 (045.6)
31.3 (096.3)
0.11

17.5 (0100.4)
15.1 (074.4)
0.49

46.2 (0127.2)
25.6 (461.6)
0.87

55.1 (2.2146)
67.6 (0243.9)
0.83

38.6 (090.5)
123.8 (0462.9)
0.22

116.1 (15.6306.3)
110.8 (18.5281)
0.95

144 (14.2387.6)
124 (0314.9)
0.95

132.5 (0279.4)
201.1 (0770.5)
0.88

117.3 (20.8305.1)
208.9 (25.4481.5)
0.1

25.2 (0111.2)
30.3 (099.8)
0.68

16.3 (061.6)
22.7 (085.2)
0.81

12.8 (060.5)
14.4 (0101.4)
0.97

18.7 (070.1)
44.3 (0204.8)
0.73

1.6 (010.1)
14.01 (046.4)
0.008*

6.2 (020.8)
8.8 (042.1)
1

11.1 (035.8)
17.2 (098.8)
0.93

0.9 (05.8)
47.8 (0260.8)
0.038*

81.6 (11.9136)
102.6 (54.2268.5)
0.41

49.8 (0147.4)
77 (39.6137.8)
0.04*

61.6 (4.1212.7)
67 (9.3252.1)
0.93

34.7 (0101.5)
94.9 (9.3317.5)
0.1

227.9 (58.5530.9)
213.2 (113.6352.9)
0.58

110.3 (13.8385.2)
208.5 (40364.3)
0.035*

142.8 (0576.7)
208.7 (0899,1)
0.5

79.1 (17.2167.4)
261.4 (38.21109.5)
0.08

46.6 (0334.9)
36.7 (0238.3)
0.97

86.4 (0272.3)
48.6 (0175.6)
0.26

103.6 (0344.5)
278.8 (03279.9)
0.49

23.2 (088.8)
104.3 (0511.8)
0.27

109.3 (0608)
162.3 (0693.2)
0.37

197.2 (0797.4)
310.5 (01338.5)
0.86

44.6 (02153.8)
521.6 (02737.8)
92

73.8 (0496.7)
71.5 (0290.8)
1

Results are expressed as median and minimummaximum values in parentheses (MannWhitney test, *P < 0.05).

chemoradiotherapy-induced OM, whereas reduced in

patients undergoing HSCT. The most significant inflammatory mediator was IL-6 levels that followed the clinical
course of OM in both study of Silva et al. [25] as in the
present study and may contribute with inflammation and
OM severity.
Studies of patients with OM undergoing RT showed an
increase in IL-6 and TNF-a concentrations [33] and a
decrease in salivary EGF levels [28,32] with increasing
radiation doses. In addition, serum IL-6 levels in patients
who had received combined chemoradiotherapy were
found a positive correlation with OM severity [26]. In

this context, studies must be undertaken based on the

pathogenesis of mucositis in order to establish the best
treatment for the patient.
LLLT has been considered an important tool for the
prevention and treatment of the most debilitating
complication of chemoradiotherapy for head and neck
cancer, as it reduces the severity of the OM [1420]. This
finding was also observed in present clinical trial and
consequently contributes with a reduction in the interruption of treatment. One possible mechanism for
reducing OM severity could be the anti-inflammatory
and analgesic effect of the laser irradiation on the local

302

OTON-LEITE ET AL.

tissue. However, the mechanisms underlying the antiinflammatory and analgesic properties of low-level laser
remain unknown.
Experimental studies have shown that, in both in vitro
and in vivo conditions, LLLT can modulate the responses of
tissue repair [3440], as well as the levels of proinflammatory and anti-inflammatory cytokines [4146].
To date, no clinical trials have investigated the biologic
modulation of inflammatory mediators by LLLT in the
prevention or treatment of chemoradiotherapy-induced
OM. Only study of Silva et al. [25] investigated the effect of
LLLT in chemotherapy-induced OM in patients submitted
to transplantation and demonstrated a significant reduction in the severity of OM in laser group. A higher
concentration of IL-10 levels was observed in blood plasma
and saliva and MMP2 levels in saliva on D 7 when
compared to the control group, suggesting that the high
level of IL-10 in saliva may have contributed to a reduction
in local inflammation and the further increase in MMP-2
level on D 7 in response to LLLT may also have
contributed to the healing process. However, the authors
suggested that patients with different underlying disease
and receiving different conditioning regimen may have
hindered the evaluation of LLLT effect on inflammation in
this study.
Our study showed that after 21 sessions of LLLT, there
was a significant reduction of the severity of OM in laser
group compared with control, which reflected biologically
in a significant reduction of IL-6 and FGF at the end of RT.
In addition, patients undergoing LLLT showed a slightly
lower concentration of IL-1b, TNF-a, MMPs, EGF, and
VEGF when compared to the control group at most of the
times evaluated. These findings allow one to hypothesize
that LLLT contributes to the development of a less
exacerbated inflammatory response in patients submitted
to it during chemoradiation for head and neck cancer.
Studies which evaluated the effect of LLLT in experimental models of OM induced by 5-FU in hamsters have
shown that it reduced the severity of mucositis and would
seem to be related to the reduction of COX-2 level and
neutrophil infiltration which contributes to the decrease in
the inflammatory process [37,38]. In addition, studies in
animal models with other diseases such as tendinitis [34,44], osteoarthritis [45], and acute inflammation [41,42,46] found that LLLT reduced the migration of
inflammatory cells [34,45], TNF-a [4143,45], COX-2 [44],
IL-1b [39,42,4446], and IL-6 concentrations [4246],
which contributes to an overall attenuation in inflammatory response.
One interesting finding in this study was that IL-6
concentrations were lower in the laser group than in the
control group throughout the treatment. This particular
fact shows that this inflammatory mediator may play an
important role in reducing the severity of OM in the laser
group. The IL-6, despite a wide range of biological
activities, acts on phagocyte migration and other inflammatory cells, increases vascular permeability, stimulates
tissue degradation by activating matrix metalloproteinases which culminate in tissue injury [43,44]. Therefore,

lower levels of IL-6 would suggest less damage to the oral

mucosa.
Furthermore, the significant reduction in IL-6 concentration at the end of RT (after 21 sessions of laser therapy)
suggests that LLLT could possibly have a cumulative
effect. NG et al. [47] suggested that multiple sessions of
laser therapy is crucial to improve the tissue repair after
injury in animals. In accordance with the laser therapy
protocol recommended by Sim~oes et al. [18] for patients
undergoing RT to treat head and neck cancers, we were
able to show a beneficial effect of LLLT performed three
times a week with a dose of 6 J/cm2. However, future
studies are needed considering different LLLT parameters
such as repeated daily treatment sessions in order to verify
the possible biomodulation induced by laser therapy. In
addition, recent studies showed that application of LLLT to
OM lesions located close to head and neck tumors may
favor tumor growth if malignant cells are present in the
irradiation field [48,49]. For this reason, in our study, the
laser irradiation was not performed in the surgical bed of
the tumoral area removed previously. Further investigations about the impact of LLLT on tumoral microenvironment should be conducted.
The less exacerbated inflammation process in patients
undergoing LLLT showed a tendency toward lower
concentrations of IL-1, TNF-a, and MMPs possibly
reflected in lower concentrations of FGF, EGF, and
VEGF especially at the beginning and end of treatment.
These findings suggest that the healing process was less
required in patients undergoing LLLT. In consonance with
our findings, in an experimental model of OM in hamsters,
Lopes et al. [37] showed that LLLT with output power of
100 mW was associated with a decrease in VEGF levels
when compared to the control group. In the first weeks of
RT, Epstein et al. [32] showed a reduction in EGF levels
which remained reduced throughout the treatment. On the
other hand, experimental studies have shown an increase
in the production of FGF and VEGF levels as an effect of
photobiomodulation [50,51].
The wound healing process is a complex series of events
regulated by growth factors, cytokines, and extracellular
matrix elements [2]. In our study, in control group MMPs
levels gradually increased with an increase in OM severity.
However, a slight decrease in MMP levels was found in the
laser group throughout the treatment. Experimental
studies in rats have shown that the production of MMP2 [36,51] and MMP-9 were higher in the injury group than in
the laser group [36]. There is limited evidence in the
literature with regard to MMP levels in the pathogenesis of
mucositis [11]. Al Dasooqi et al. [11] suggest that patients
with mucositis showed a significant increase in MMP levels.
Therefore, a tendency toward a lower concentration of
MMPs in the laser group would suggest that LLLT can help
reduce damage in OM with less intense tissue degradation.
A slight reduction in anti-inflammatory cytokines
(TGF-b and IL-10) was observed in the laser group
when compared with the control group at almost all
evaluated times. Pires et al. [44] suggested that a
reduction in TGF-b induced by laser may be an indirect

EFFECT OF LOW-LEVEL LASER THERAPY IN CANCER PATIENTS

response to reduction in pro-inflammatory cytokines. In

this same context, Citrin et al. [33] showed a decrease in
salivary IL-10 levels associated with lower grades of OM
in patients receiving chemoradiation without LLLT for
head and neck malignancies. Another important point
noted by Citrin et al. [33] which could also be gleaned
from our results is that the sampling of salivary cytokine
levels in patients undergoing RT for head and neck
tumors is feasible and may provide a noninvasive
biomarker of local effects of irradiation.
Although this clinical trial selected patients with similar
treatment protocols for chemotherapy and RT who had also
received previous dental and hygiene care, some limitations can be listed. Other conditions such as tumor stage,
oral diseases, nutritional status, bacterial colonization,
effects of chemoradiation on salivary glands, and individual genetic influence could explain the differences in the
inflammatory response and repair process, which could
explain the great variability found in the concentration of
inflammatory mediators from one patient to another in this
study. Future studies are needed including larger number
of patients for reducing data variability and with more
specific inclusion and exclusion criteria for controlling
potential confounders.
In conclusion, despite its limitations, this trial provides
evidence that LLLT brought about a clinical improvement in
OM in head and neck cancer patients undergoing chemoradiotherapy. This resulted in the attenuation of the
inflammatory process and less required repair. However, it
was not possible to establish completely the exact action
mechanism of LLLT in OM due to the fact that it is a dynamic
process involving biological events influenced by the oral
micro-environment. Therefore, additional studies with large
numbers of subjects and different protocols of laser irradiation are warranted in order to understand the effect of LLLT
in the control of chemoradiotherapy-induced OM.

5.
6.

7.

8.

9.

10.

11.

12.

13.

14.

ACKNOWLEDGMENTS
This study was supported by grants from the National
Council for Scientific and Technological Development
(CNPq grantsprocess number 479587/2010-8) and the
Foundation for Research Support in the State of Goi

as
(FAPEGgrant 2010102670007). The authors thank the
patients of Ara

ujo Jorge Hospital, Association of Cancer
Combat of Goi

as, Goi^
ania, Brazil.

15.

16.

REFERENCES
1. Sonis ST. The pathobiology of mucositis. Nat Rev Cancer
2004;4(4):277284.
2. Sonis ST, Elting LS, Keefe D, Peterson DE, Schubert M,
Hauer-Jensen M, Bekele BN, Raber-Durlacher J, Donnelly
JP, Rubenstein EB. Perspectives on cancer therapy-induced
mucosal injury: pathogenesis, measurement, epidemiology,
and consequences for patients. Cancer 2004;100(9 Suppl):
19952025.
3. Rosenthal DI. Consequences of mucositis-induced treatment
breaks and dose reductions on head and neck cancer
treatment outcomes. J Support Oncol 2007;5(9 Suppl 4):
2331.
4. Elting LS, Keefe DM, Sonis ST, Garden AS, Spijkervet FK,
Barasch A, Tishler RB, Canty TP, Kudrimoti MK, VeraLlonch M. Patient-reported measurements of oral mucositis

17.

18.

19.

303

in head and neck cancer patients treated with radiotherapy

with or without chemotherapy: Demonstration of increased
frequency, severity, resistance to palliation, and impact on
quality of life. Cancer 2008;113(10):27042713.
Russo G, Haddad R, Posner M, Machtay M. Radiation
treatment breaks and ulcerative mucositis in head and neck
cancer. Oncologist 2008;13(8):886898.
Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D,
Haimovitz-Friedman A, Cordon-Cardo C, Kolesnick R.
Endothelial apoptosis as the primary lesion initiating
intestinal radiation damage in mice. Science 2001;293(5528):293297.
Logan RM, Stringer AM, Bowen JM, Yeoh AS, Gibson RJ,
Sonis ST, Keefe DM. The role of pro-inflammatory cytokines
in cancer treatment-induced alimentary tract mucositis:
pathobiology, animal models and cytotoxic drugs. Cancer
Treat Rev 2007;33(5):448460.
Ong ZY, Gibson RJ, Bowen JM, Stringer AM, Darby JM,
Logan RM, Yeoh AS, Keefe DM. Pro-inflammatory cytokines
play a key role in the development of radiotherapy-induced
gastrointestinal mucositis. Radiat Oncol 2010;5:22.
Stringer AM, Gibson RJ, Bowen JM, Logan RM, Yeoh AS,
Keefe DM. Chemotherapy-induced mucositis: The role of
gastrointestinal microflora and mucins in the luminal
environment. J Support Oncol 2007;5(6):259267.
Al-Dasooqi N, Gibson RJ, Bowen JM, Logan RM, Stringer AM,
Keefe DM. Matrix metalloproteinases are possible mediators for the development of alimentary tract mucositis in
the dark agouti rat. Exp Biol Med 2010;235(10):1244
1256.
Al-Dasooqi N, Gibson RJ, Bowen JM, Keefe DM. Matrix
metalloproteinases: Key regulators in the pathogenesis of
chemotherapy-induced mucositis?. Cancer Chemother Pharmacol 2009;64(1):19.
Jensen SB, Jarvis V, Zadik Y, Barasch A, Ariyawardana A,
Hovan A, Yarom N, Lalla RV, Bowen J, Elad S. Systematic
review of miscellaneous agents for the management of oral
mucositis in cancer patients. Support Care Cancer 2013;21(11):32233232.
Saunders DP, Epstein JB, Elad S, Allemano J, Bossi P, van
de Wetering MD, Rao NG, Potting C, Cheng KK, Freidank
A, Brennan MT, Bowen J, Dennis K, Lalla RV. Systematic
review of antimicrobials, mucosal coating agents, anesthetics, and analgesics for the management of oral mucositis
in cancer patients. Support Care Cancer 2013;21(11):
31913207.
Antunes HS, Herchenhorn D, Small IA, Ara

ujo CM, Vi

egas
CM, Cabral E, Rampini E, Rodrigues PC, Silva TG, Ferreira
EM, Dias FL, Ferreira CG. Phase III trial of low-level laser
therapy to prevent oral mucositis in head and neck cancer
patients treated with concurrent chemoradiation. Radiother
Oncol 2013;9(2):297302.
Migliorati C, Hewson I, Lalla RV, Antunes HS, Estilo CL,
Hodgson B, Lopes NN, Schubert MM, Bowen J, Elad S.
Systematic review of laser and other light therapy for the
management of oral mucositis in cancer patients. Support
Care Cancer 2013;21(1):333341.
Gautam AP, Fernandes DJ, Vidyasagar MS, Maiya GA. Low
level helium neon laser therapy for chemoradiotherapy
induced oral mucositis in oral cancer patientsA randomized
controlled trial. Oral Oncol 2012;48(9):893897.
Carvalho PA, Jaguar GC, Pellizzon AC, Prado JD, Lopes RN,
Alves FA. Evaluation of low-level laser therapy in the
prevention and treatment of radiation-induced mucositis: A
double-blind randomized study in head and neck cancer
patients. Oral Oncol 2011;47(12):11761181.
Sim~
oes A, Eduardo FP, Luiz AC, Campos L, S

a PH, Crist

ofaro
M, Marques MM, Eduardo CP. Laser phototherapy as topical
prophylaxis against head and neck cancer radiotherapyinduced oral mucositis: Comparison between low and high/
low power lasers. Lasers Surg Med 2009;41(4):264270.
Oton-Leite AF, Corr^
ea de Castro AC, Morais MO, Pinezi JC,
Leles CR, MendonSc a EF. Effect of intraoral low-level laser
therapy on quality of life of patients with head and neck
cancer undergoing radiotherapy. Head Neck 2012;34(3):
398404.

304

OTON-LEITE ET AL.

20. Oton-Leite AF, Elias LS, Morais MO, Pinezi JC, Leles CR,
Silva MA, MendonSc a EF. Effect of low level laser therapy in
the reduction of oral complications in patients with cancer of
the head and neck submitted to radiotherapy. Spec Care
Dentist 2013;33(6):294300.
21. Silva GB, MendonSc a EF, Bariani C, Antunes HS, Silva MA.
The prevention of induced oral mucositis with low-level
laser therapy in bone marrow transplantation patients: A
randomized clinical trial. Photomed Laser Surg 2011;29(1):2731.
22. Schubert MM, Eduardo FP, Guthrie KA, Franquin JC,
Bensadoun RJ, Migliorati CA, Lloid CM, Eduardo CP, Walter
NF, Marques MM, Hamdi M. A phase III randomized doubleblind placebo-controlled clinical trial to determine the efficacy
of low level laser therapy for the prevention of oral mucositis
in patients undergoing hematopoietic cell transplantation.
Support Care Cancer 2007;15(10):11451154.
23. Lalla RV, Bowen J, Barasch A, Elting L, Epstein J,
Keefe DM, McGuire DB, Migliorati C, Nicolatou-Galitis O,
Peterson DE, Raber-Durlacher JE, Sonis ST, Elad S.
Mucositis Guidelines Leadership Group of the Multinational Association of Supportive Care in Cancer and International Society of Oral Oncology (MASCC/ISOO). MASCC/
ISOO clinical practice guidelines for the management
of mucositis secondary to cancer therapy. Cancer 2014;
120(10):14531461.
24. Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci
1993;694:7277.
25. Silva GB, Sacono NT, Othon-Leite AF, MendonSc a EF, Arantes
AM, Bariani C, Duarte LG, Abreu MH, Queiroz-J

unior CM,
Silva TA, Batista AC. Effect of low-level laser therapy on
inflammatory mediator release during chemotherapy-induced oral mucositis: A randomized preliminary study.
Lasers Med Sci 2015;30(1):117126.
26. Meirovitz A, Kuten M, Billan S, Abdah-Bortnyak R, Sharon A,
Peretz T, Sela M, Schaffer M, Barak V. Cytokines
levels, severity of acute mucositis and the need of PEG
tube installation during chemo-radiation for head and
neck cancerA prospective pilot study. Radiat Oncol
2010;5:16.
27. Shoval I, Kushner JA, Sukhu B, Wood R, Kiss T, Lawrence
HP, Tenenbaum HC. The relationship between mouthrinse
matrix metalloproteinases (MMP-1, 8, 13) and albumin
levels with the degree of oral mucositis in allogeneic stem
cell transplant patients. Bone Marrow Transpl 2005;8(13):
3338.
28. Dumbrigue HB, Sandow PL, Nguyen KH, Humphreys-Beher
MG. Salivary epidermal growth factor levels decrease in
patients receiving radiation therapy to the head and neck.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;89(6):710716.
29. Lalla RV, Pilbeam CC, Walsh SJ, Sonis ST, Keefe DM,
Peterson DE. Role of the cyclooxygenase pathway in
chemotherapy-induced oral mucositis: A pilot study. Support
Care Cancer 2010;18(1):95103.
30. Fall-Dickson JM, Ramsay ES, Castro K, Woltz P, Sport

es C.
Oral mucositis-related oropharyngeal pain and correlative
tumor necrosis factor-alpha expression in adult oncology
patients undergoing hematopoietic stem cell transplantation.
Clin Ther 2007;29 Suppl:25472561.
31. Morales-Rojas T, Viera N, Mor

on-Medina A, Alvarez CJ,
Alvarez A. Proinflammatory cytokines during the
initial phase of oral mucositis in patients with acute
lymphoblastic leukaemia. Int J Paediatr Dent 2012;22(3):191196.
32. Epstein JB, Gorsky M, Guglietta A, Le N, Sonis ST. The
correlation between epidermal growth factor levels in saliva
and the severity of oral mucositis during oropharyngeal
radiation therapy. Cancer 2000;89(11):22582265.
33. Citrin DE, Hitchcock YJ, Chung EJ, Frandsen J, Urick ME,
Shield W, Gaffney D. Determination of cytokine protein
levels in oral secretions in patients undergoing radiotherapy
for head and neck malignancies. Radiat Oncol 2012;
7:64.
34. Casalechi HL, de Farias Marques AC, da Silva EA, Aimbire F,
Marcos RL, Lopes-Martins RA, de Carvalho P de T, Albertini

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

R. Analysis of the effect of phototherapy in model with

traumatic Achilles tendon injury in rats. Lasers Med Sci
2014;29(3):10751081.
Usumez A, Cengiz B, Oztuzcu S, Demir T, Aras MH,
Gutknecht N. Effects of laser irradiation at different wavelengths (660, 810, 980, and 1,064 nm) on mucositis in an
animal model of wound healing. Lasers Med Sci 2014;29(6):18071813.
Alves AC, Albertini R, dos Santos SA, Leal-Junior EC,
Santana E, Serra AJ, Silva JA Jr, de Carvalho P de T. Effect
of low-level laser therapy on metalloproteinase MMP-2 and
MMP-9 production and percentage of collagen types I and III
in a papain cartilage injury model. Lasers Med Sci 2014;29(3):911919.
Lopes NN, Plapler H, Chavantes MC, Lalla RV, Yoshimura
EM, Alves MT. Cyclooxygenase-2 and vascular endothelial
growth factor expression in 5-fluorouracil-induced oral
mucositis in hamsters: Evaluation of two low-intensity
laser protocols. Support Care Cancer 2009;17(11):1409
1415.
Lopes NN, Plapler H, Lalla RV, Chavantes MC, Yoshimura
EM, da Silva MA, Alves MT. Effects of low-level laser therapy
on collagen expression and neutrophil infiltrate in 5fluorouracil-induced oral mucositis in hamsters. Lasers
Surg Med 2010;42(6):546552.
Safavi SM, Kazemi B, Esmaeili M, Fallah A, Modarresi A, Mir
M. Effects of low-level He-Ne laser irradiation on the gene
expression of IL-1beta, TNF-alpha, IFN-gamma, TGF-beta,
bFGF, and PDGF in rats gingival. Lasers Med Sci 2008;23(3):331335.
Bjordal JM, Lopes-Martins RA, Iversen VV. A randomised,
placebo controlled trial of low level laser therapy for activated
Achilles tendinitis with microdialysis measurement of peritendinous prostaglandin E2 concentrations. Br J Sports Med
2006;40(1):7680.
Aimbire F, Albertini R, Pacheco MT, Castro-Faria-Neto HC,
Leonardo PS, Iversen VV, Lopes-Martins RA, Bjordal JM.
Low-level laser therapy induces dose-dependent reduction of
TNF-alpha levels in acute inflammation. Photomed Laser
Surg 2006;24(1):3337.
Albertini R, Villaverde AB, Aimbire F, Bjordal J, Brugnera A,
Mittmann J, Silva JA, Costa M. Cytokine mRNA expression is
decreased in the subplantar muscle of rat paw subjected to
carrageenan-induced inflammation after low-level laser
therapy. Photomed Laser Surg 2008;26(1):1924.
Fukuda TY, Tanji MM, Silva SR, Sato MN, Plapler H.
Infrared low-level diode laser on inflammatory process
modulation in mice: Pro- and anti-inflammatory cytokines.
Lasers Med Sci 2013;28(5):13051313.
Pires D, Xavier M, Ara

ujo T, Silva JA Jr, Albertini F. Lowlevel laser therapy (LLLT; 780nm) acts differently on mRNA
expression of anti- and pro-inflammatory mediators in an
experimental model of collagenase-induced tendinitis in rat.
Lasers Med Sci 2011;26(1):8594.
Alves AC, Vieira R, Leal-Junior E, dos Santos S, Ligeiro AP,
Albertini R, Junior J, de Carvalho P. Effect of low-level laser
therapy on the expression of inflammatory mediators and on
neutrophils and macrophages in acute joint inflammation.
Arthritis Res Ther 2013;15(5):R116.
Dos Santos SA, Alves AC, Leal-Junior EC, Albertini R, Vieira
Rde P, Ligeiro AP, Junior JA, De Carvalho P de T.
Comparative analysis of two low-level laser doses on the
expression of inflammatory mediators and on neutrophils and
macrophages in acute joint inflammation. Lasers Med Sci
2014;29(3):10511058.
Ng GY, Fung DT, Leung MC, Guo X. Comparison of single
and multiple applications of GaAlAs laser on rat medial
collateral ligament repair. Lasers Surg Med 2004;34(3):
285289.
de C Monteiro JS, Pinheiro AN, de Oliveira SC, Aciole GT,
Sousa JA, Canguss MC, Dos Santos JN. Influence of laser
phototherapy (l660nm) on the outcome of oral chemical
carcinogenesis on the hamster cheek pouch model: Histological study. Photomed Laser Surg 2011;29(11):741745.


Gomes Henriques AC,
Ginani F, Oliveira RM, Keesen TS,
Galv~
ao Barboza CA, Oliveira Rocha HA, de Castro JF,

EFFECT OF LOW-LEVEL LASER THERAPY IN CANCER PATIENTS

Della Coletta R, de Almeida Freitas R. Low-level laser
therapy promotes proliferation and invasion of oral squamous
cell carcinoma cells. Lasers Med Sci 2014;29(4):1385
1395.
50. Saygun I, Karacay S, Serdar M, Ural AU, Sencimen M, Kurtis
B. Effects of laser irradiation on the release of basic fibroblast
growth factor (bFGF), insulin like growth factor-1 (IGF-1),

305

and receptor of IGF-1 (IGFBP3) from gingival fibroblasts.

Lasers Med Sci 2008;23(2):211215.
51. Cury V, Moretti AI, Assis L, Bossini P, Crusca Jde S, Neto CB,
Fangel R, de Souza HP, Hamblin MR, Parizotto NA. Low level
laser therapy increases angiogenesis in a model of ischemic
skin flap in rats mediated by VEGF, HIF-1a and MMP-2.
Photochem Photobiol B 2013;125:164170.