Nearly all patients with advanced head and neck cancer (HNC) suffer complications from treatment with radiation therapy (RT) or chemoradiotherapy (CRT). CRT is currently the standard of care with or without surgery in advanced HNC. Typically, there is an increased frequency and severity of side effects, particularly when chemotherapy (CT) is combined with accelerated or hyperfractionated RT regimens. It is now recognized that organ preservation in HNC treatment is not synonymous with function preservation, and effects on quality of life (QoL) must be considered in cancer treatment planning and extending survival [2, 3].
RT to the head and neck, with or without CT, damages adjacent tissues within the radiation field despite continuing efforts to minimize these effects. Furthermore, targeted therapies administered as single agents, and combined with RT or CRT, may generate additional symptoms [4-6]. Acute complications in the orofacial region and neck include oral mucositis (OM), pain, dysphagia, infections, salivary changes, dysgeusia, and dermatitis. Common chronic complications are hyposalivation and xerostomia, mucosal infections, mucosal atrophy, neuropathies including mucosal pain, dysgeusia, tooth decalcification and rampant caries, progression of periodontitis, soft tissue and/or bone necrosis, mucocutaneous and muscular fibrosis, dysphagia, trismus, lymphedema, dermatitis, and voice and speech alterations. The severity of complications varies depending upon the type and site of the tumor, mode and intensity of therapies involved, and individual patient characteristics. Orofacial and neck complications are associated with morbidity and mortality, increased use of health care resources and costs, and may compromise patient adherence to cancer therapy protocols resulting in suboptimal outcomes. Most patients develop multiple complications, which result in a significant burden of illness with negative impact on QoL[1, 7-9].
Supportive care addressing these complications must continue from initial diagnosis of HNC, through treatment and survival. However, many interventions have limitations and are primarily palliative in nature.
Among the presently available supportive care measures, low level laser therapy (LLLT)/photobiomodulation (BPM) has shown significant promise. LLLT/PBM refers to light therapy that may control pain, stimulate tissue regeneration and reduce inflammation. These treatments were originally referred to as “low level laser” because the light is of low intensity compared with other forms of medical laser treatment, which are used for ablation, cutting, and coagulation. However, at the 2014 joint North American Association for Laser Therapy (NAALT) & World Association for Laser Therapy (WALT) conference a consensus committee convened to discuss the diverse nomenclature, false positive database results and inappropriate emphasis on the word “laser”. PBM was accepted as the preferred name with the following definition: “The therapeutic use of light [e.g. visible, near infrared (NIR), infrared (IR)] absorbed by endogenous chromophores, triggering non-thermal, non-cytotoxic, biological reactions through photochemical or photophysical events, leading to physiological changes”.
These photobiological reactions have been shown to occur in various tissues (skin, mucosa, muscle, tendon, ligament, cartilage, bone, nerve, lymph, blood components and blood vessels). Systematic reviews have suggested efficacy of LLLT/PBM for OM management in hematopoietic stem cell transplant (HSCT) recipients and in HNC patients [11-16]. Whereas in most studies LLLT/PBM is applied intra-orally on the oral mucosal tissues, studies indicate that it may also be administered extra-orally, with a resultant effect on structures at risk for OM transcutaneously, thereby enhancing the ease of delivery and possibly the efficacy of treatment [14, 17]. In addition, new generation LLLT/PBM devices consisting of a cluster of laser or light-emitting diode (LED) beams, instead of a single laser point, provide exposure of larger fields and when used with appropriate parameters, the light is able to penetrate into tissues sufficiently to activate cellular processes . This finding suggests that extra-oral administration of LLLT/PBM (with or without concurrent use of intra-orally administered LLLT/PBM), and taking advantage of advances in LLLT/PBM technology enables the light to reach other anatomical structures of the head and neck at risk for RT and CRT-induced complications. This may broaden the range of indications for PBM for the prevention and treatment of cancer treatment-induced complications.
A task force consisting of an international multidisciplinary panel of clinicians and researchers with expertise in the area of supportive care in cancer and/or LLLT/PBM clinical application and dosimetry was formed. The mission of this group is to aid in the design of LLLT/PBM study protocols, identify validated outcome measures, and test the efficacy and safety of proposed protocols for the management of complications related to cancer therapy.
The goals of the present paper are to: i) discuss LLLT/PBM mechanisms and safety issues; ii) identify selected oral, oropharyngeal, facial, and neck complications of treatment for HNC, in which LLLT/PBM may have potential for prophylaxis and/or treatment; iii) propose LLLT/PBM parameters for prophylaxis and therapy to mitigate these complications based on current evidence and knowledge, and iv) discuss directions of future research related to the use of LLLT/PBM in HNC.
LLLT/PBM therapeutic effects
Mechanisms of action
LLLT/PBM has been consistently shown in laboratory studies to have distinct biological effects, and has a dose-dependent mechanism of action at the cellular level[19, 20]. Since the introduction of LLLT/PBM in 1967, over 400 randomized, double-blinded (some placebo-controlled) clinical trials have been published for multiple applications. The first clinical application of LLLT/PBM was for enhancement of wound healing . A meta-analysis including animal and human studies concluded that LLLT/PBM was an effective tool for accelerating wound repair and tissue regeneration . It has been shown that LLLT/PBM influences different phases of wound healing including: i) the inflammatory phase, in which immune cells migrate to the site of tissue injury, ii) the proliferative phase, which includes stimulation of fibroblasts and macrophages as well as other repair components, and iii) the remodeling phase, consisting of collagen deposition and rebuilding of the extracellular matrix at the wound site .
Although the complex biological mechanisms underlying the therapeutic effects of LLLT/PBM have not been completely elucidated and may vary among different cell types and tissue states (healthy versus stressed or hypoxic), laboratory and clinical studies suggest that LLLT/PBM significantly reduces inflammation and prevents fibrosis [24-29]. Moreover, LLLT/PBM, when delivered appropriately, reduces pain and improves optimal function [19, 30-32]. In addition, in vivo studies show that LLLT/PBM is neuroprotective and may benefit neurodegenerative diseases and neurotrauma [33, 34].
Current data suggest that LLLT/PBM acts predominantly on cytochrome c oxidase (CcO) in the mitochondrial respiratory chain by facilitating electron transport resulting in an increased transmembrane proton gradient that drives adenosine triphosphate (ATP) production . ATP is the universal energy source in living cells essential for all biologic reactions, and even a small increase in ATP levels can enhance bioavailability to power the functions of cellular metabolism . In addition, the absorption of red or NIR light may cause a short, transient burst of reactive oxygen species (ROS) that is followed by an adaptive reduction in oxidative stress.
A low concentration of ROS activates many cellular processes, since ROS activates transcription factors including nuclear factor kappa B (NF-κB), resulting in the upregulation of stimulatory and protective genes . These genes generate growth factors belonging to the fibroblast growth factor family, cytokines, and chemokines that are involved in tissue repair.
In hypoxic or otherwise stressed cells, mitochondria produce nitric oxide (mtNO), which binds to CcO and displaces oxygen. This binding results in inhibition of cellular respiration, decreased ATP production and increased oxidative stress (a state that develops when the levels of ROS exceed the defense mechanisms), leading to the activation of intracellular signaling pathways, including several transcription factors . These include redox factor-1 (Ref-1), activator protein-1 (AP-1), NF-κB, p53, activating transcription factor/cAMP-response element–binding protein (ATF/ CREB), hypoxia-inducible factor (HIF)-1, and HIF-like factor . These transcription factors induce down-stream production of both inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), Interleukin [IL]-1 and IL-6, cyclooxygenase (COX)-2, and prostaglandin E2 (PGE-2)[39, 41, 42] and anti-inflammatory mediators [Transforming growth factor (TGF)-beta, IL-10]. There is evidence suggesting that when LLLT/PBM is administered with appropriate parameters to stressed cells, NO is dissociated from its competitive binding to CcO, ATP production is increased, and the balance between pro- and antioxidant mediators is restored, resulting in a reduction of oxidative stress. For example, LLLT/PBM has been shown to attenuate the production of ROS by human neutrophils . Silveira et al  reported that LLLT/PBM reduced ROS in an animal model of traumatic tissue injury; whereas a study in a model of acute lung inflammation found LLLT/PBM to reduce the generation of TNF-α and to increase IL-10 . In addition, NO is a potent vasodilator  and can increase the blood-supply to the laser-illuminated tissue. LLLT-mediated vascular regulation increases tissue oxygenation and also allows for greater traffic of immune cells, which may contribute to the promotion of wound repair and regeneration.
Analgesic effects are probably induced by additional mechanisms rather than by the increased ATP/reduced oxidative stress model. LLLT/PBM with a relatively high power density (> 300 mW/cm2), when absorbed by nociceptors, has an inhibitory effect on A and C neuronal pain fibers. This slows neural conduction velocity, reduces amplitude of compound action potentials and suppresses neurogenic inflammation .
Virtually all conditions modulated by LLLT/PBM (e.g., ulceration, inflammation, edema, pain, fibrosis, neurological and muscular injury) are thought to be involved in the pathogenesis of (C)RT-induced complications in patients treated for HNC. For example, in an animal model of OM it was demonstrated that LLLT/PBM decreased COX-2 expression  and decreased the number of neutrophils in the inflammatory infiltrate . Moreover, in the chronic sequelae of (C)RT an excessive fibroblastic response is hypothesized to be related to acute oxidative injury, with resulting cell damage, ischemia, and an ongoing inflammatory response resulting in fibrosis . The critical difference between normal wound healing and fibrosis development appears to be, that in fibrosis, signaling pathways escape normal cellular regulation . Reduction of fibrosis could be mediated by the beneficial effects of LLLT/PBM on the oxidant/antioxidant balance , down regulation of TGF-β, and inhibition of excessive fibroblast proliferation .
Although most studies have demonstrated efficacy in management of both acutely and chronically affected tissues, not all LLLT/PBM investigations have yielded positive outcomes. As discussed below, these divergent results may be attributed to several factors, including dosimetry. It has been observed that increasing the overall dose of LLLT/PBM may have a counter-productive effect compared with the benefit obtained with lower doses .
LLLT/PBM parameters have been mostly reported within the red and NIR wavelength range of 600 nanometer (nm) – 1,000 nm, with a power density of between 5 milliwatts (mW)/cm2- 150 mW/cm2 and are typically applied for 30 – 60 seconds per point. The therapeutic effect is anticipated to be dictated by the energy density measured in J/cm2. Evidence can be found in the literature for parameters as widely divergent as 0.1 to 12 J/cm2. Commonly reported LLLT/BPM devices include helium-neon (HeNe) gas laser, gallium–arsenide (GaAs), neodymium-doped yttrium aluminum garnet (Nd:YAG), gallium aluminum arsenide (GaAlAs), Indium gallium aluminum phosphide (InGaAlP) diode lasers, non-thermal, non-ablative carbon dioxide (CO2) lasers, and LED arrays.
The PBM effects on the exposed tissues depend on: cell type, redox state of the cell, irradiation parameters (wavelength, power density), and time of exposure [14, 54]. A biphasic dose response has been shown, which underlines that there are optimal irradiation and dose parameters, although these will vary according to the depth of the pathology below the mucosal surface or skin. Doses lower than the optimal value, may have a diminished effect, while doses higher than optimal can have negative therapeutic outcomes [39, 54].
Thus for LLLT/PBM to be effective, the irradiation parameters, including the energy delivered, power density, pulse structure, delivery to the appropriate anatomical location, appropriate treatment timing and repetition, need to be within the biostimulatory dose windows [13, 39, 54-56].
For example, a study in which LLLT/PBM was not found effective in reducing severe OM in HNC patients treated with CRT may have used too low dosing .
Titrating adequate doses and defining the other required laser parameters according to evidence gathered in a systematic way for each indication is a prerequisite for a successful use of this technique. Without standardization in beam measurement, dose calculation, and the correct reporting of these parameters, studies will not be reproducible, and outcomes will not be consistent. A common misconception is that wavelength and energy (in J) or energy density (J/cm2) are all that is necessary in order to replicate a successful treatment, and that it does not matter what the original power, power density, and duration parameters are [58, 59].
A checklist to help researchers understand and report all the necessary parameters for a reproducible scientific study has been developed (Table 1) . It is not uncommon to find discrepancies between the specifications provided by a device manufacturer and the actual performance of the device . Therefore, device maintenance including power measurements should be carried out regularly during research trials and also in clinical practice.
Potential effects of LLLT/PBM on tumors
Since its first use clinically in the late 1960’s, the potential for LLLT/BPM to impact cancer risk or development has been studied. However, the potential effects of LLLT/BPM on tumor, including tumor protection, tumor promotion, no (direct) effects or beneficial effects, require ongoing studies before definitive conclusions can be made.
A vast amount of progress has been made in the past decade to advance our understanding of the molecular biology which drives squamous cell carcinoma of the head and neck (HNSCC) and the mechanism of action of LLLT/PBM. Activation of the PI3K/AKT/mTOR pathway is associated with many of the activities that may be associated with LLLT/PBM’s favorable impact on wound healing: cell survival, migration, proliferation, and angiogenesis. Yet PI3K/AKT/mTOR signaling is also among commonly dysregulated pathways associated with cancer, including HNSCC , and its activation has been reported to promote the acquisition of epithelial-mesenchymal transition, cancer stem cell phenotypes and cancer radioresistance . Conversely, inhibition of the pathway has been viewed as a potential strategy to increase radiation sensitivity of tumor cells . Recently reported data suggest that the migration of oral keratinocytes noted to occur following LLLT/BPM is attributable to activation of the AKT/mTOR signaling pathway . Consequently, the observation reported by Sperandio et al  that LLLT/BPM modified the expression of proteins related to the progression and invasion of oral cancer cell lines suggests that LLLT/PBM activation of the Akt/mTOR signaling pathway may not be desirable. The lack of data obtained from in vivo models or patients leaves open the question of the breadth of LLLT/BPM effects on malignant cells and nonmalignant tissue. For example, assuming Akt/mTOR is activated by LLLT/BPM, would tumor tissue be affected if it was distant from the site of phototherapy application, i.e. treating the mouth for OM in an individual being treated for a hypopharyngeal cancer?
TGF-β may play contradictory roles relative to tumor behavior . While, its tumor suppressive effects are notable in the early stages of carcinogenesis, it may promote growth and spread of established tumors. Through serine/threokine kinases and Smad effectors, TGF-β can act as a tumor suppressor by inhibiting proliferation and inducing apoptosis . Conversely, it may be overproduced by human tumors and is associated with induction of epithelial-mesenchymal transition, the prelude to tumor invasiveness, angiogenesis, suppression of elements of immune surveillance, and recruitment of signaling pathways that may facilitate metastases . Additionally, it appears that TGF-β1 signaling may enhance tumor progression by altering the surrounding stroma through Smad signaling . Thus, the observation that LLLT stimulates TGF-β/smad signaling pathway  could be viewed as a double-edge sword depending on when and what tissue was exposed .
Mitogen-activated protein kinase (MAPK) pathways play a significant role in cancer . Among the MAPK pathways, perhaps the best studied relative to cancer is the ERK pathway. ERK signaling is associated with a number of tumor behaviors. Of relevance to HNC is a correlation of its expression with increased epithelial growth factor receptor (EGFR) . The ERK pathway also impacts vascular epithelial growth factor (VEGF) expression and its consequent angiogenesis. While angiogenesis may be desirable from a wound healing perspective, the finding that LLLT/BPM stimulates EGFR and VEGF production through ERK signaling may be a concern in a tumor environment [74, 75].
The biological robustness of LLLT/BPM is borne out by the observations of its ability to stimulate a range of biological processes including upregulation of heat shock proteins (HSP)  and microRNAs . Relative to the current discussion, HSP is essential for cancer survival and has been identified as a potential target for anti-cancer therapy.
While the number of miRNAs that are upregulated following LLLT/BPM is substantial, of particular note is the finding that mi126 is among the list as endogenous mi126 has been reported to be associated with metastatic progression .
While the information above raises questions about possible undesirable effects of LLLT/BPM on tumor progression and response to anti-cancer treatment, some observations suggest that LLLT/BPM might favorably impact tumor behavior through its effects on vimentin expression, MyD88-dependent signaling, reduction in TLR-4 and down-regulation of NF-κB. Furthermore, upregulation of ATP signaling by LLLT/PBM may promote apoptosis, as well as differentiation of tumor cells, thereby slowing down tumor proliferation [35, 79].
The lack of consistent findings and/or the latitude of interpretation of the clinical significance of these findings hampers meaningful conclusions. The molecular mechanisms outlined above indicate the need to continue study addressing the molecular pathways affected by LLLT/BPM.
LLLT/PBM effects on tumor cell lines and data derived from in-vivo studies
The effects of LLLT/PBM on cell proliferation and differentiation have been investigated in vitro using malignant cell lines, which have generated conflicting data across a range of different tumor cell lines and LLLT/PBM parameters [80-84]. For example, Kreisler and coworkers reported proliferation of laryngeal carcinoma cells after 809 nm GaAIAs laser irradiation at energy densities between 1.96 and 7.84 J/cm2 . Werneck and coworkers also found increased cell proliferation of HEp2 carcinoma cells after LLLT/PBM exposure at different wavelengths (685 nm and 830 nm) and doses . In a study comparing LLLT/PBM administered to normal osteoblasts and to osteosarcoma cells with a range of different wavelengths and doses, only 10 J/cm2 from an 830 nm laser was able to enhance osteoblast proliferation, whereas energy densities of 1 J/cm2, 5 J/cm2, and 10 J/cm2 from a 780 nm laser decreased proliferation. Osteosarcoma cells were unaffected by 830 nm laser irradiation, whereas 670 nm laser had a mild proliferative effect . An in vitro study compared the effects of different doses of LLLT/PBM at various wavelengths on human breast carcinoma and melanoma cell lines . Although certain doses of LLLT/PBM increased breast carcinoma cell proliferation, multiple exposures had either no effect or showed negative dose response relationships. LLLT/PBM (wavelength 660nm) administered in low doses (1 J/cm2) increased in vitro proliferation and potentially increased invasive potential of tongue SCC cells . Similarly, another in vitro study suggested that LLLT/PBM (660 nm or 780 nm, 40 mW, 2.05, 3.07 or 6.15 J/cm2) may stimulate oral dysplastic and oral cancer cells to a more aggressive behavior .
In contrast, a decreased mitotic rate was found in gingival squamous cell carcinoma (SCC) after LLLT/PBM at 805 nm and energy density of 4 J/cm2 and 20 J/cm2 , whereas no effect on cell proliferation or protein expression of osteosarcoma cells was found when LLLT/PBM was administered with a wavelength of 830 nm . LLLT/PBM (808 nm; 5.85 and 7.8 J/cm2) had an inhibitory effect on the proliferation of a human hepatoma cells line  and Sroka et al  reported that glioblastoma/astrocytoma cells exhibited a slightly decreased mitotic rate after LLLT/PBM at 805 nm and 5–20 J/cm2. Similarly, 808 nm laser irradiation with an energy density of more than 5 J/cm2 inhibited cell proliferation of glioblastoma cells in vitro . Moreover, Al Watban et al  observed growth inhibition of cancer cell lines at relatively high cumulative LLLT doses. This prompted Crous and Abrahamse  to hypothesize that LLLT/PBM may have a therapeutic potential in lung cancer.
It seems unlikely that LLLT has carcinogenic effects on normal cells. The non-ionizing wavelengths of the red and NIR spectrum used in LLLT/PBM are far longer than the safety limit of 320 nm for DNA damage . No signs of malignant transformation in non-malignant epithelial cells and fibroblasts were observed following exposure to LLLT/PBM with a wavelength of 660 nm, 350 mW for 15 minutes during 3 consecutive days . In addition, no malignant transformation of normal breast epithelial cells was detected in an in vitro study comparing the effects of different doses and wavelengths of LLLT/PBM during multiple exposures .
Similarly, there are no data to suggest that LLLT/PBM may protect cancer cells against the cytotoxic effects of RT. On the contrary, Schartinger et al , who observed a pro-apoptotic effect of LLLT/PBM in HNSCC cells, whereas no anti-apoptotic effects occurred that might promote tumor cell resistance to cancer therapy. Increased apoptosis of human osteosarcoma cells was also induced by the administration of NIR (810 nm, continuous wave, 20 mW/cm2, 1.5 J/cm2) prior to NPe6-mediated photodynamic therapy as a result of increased cellular ATP and a higher uptake of the photosensitizer . Furthermore, Schaffer et al  observed that LLLT/PBM increased the loco-regional blood flow that contributed to better local oxygenation, and hypothesized that LLLT/PBM applied shortly before cancer treatment might enhance the effect of ionizing RT and local chemotherapy .
LLLT/PBM (660 nm, 30mW, 424 mW/cm2, 56.4 J/cm2, 133 sec, 4 J) applied to chemically-induced SCC in hamster cheek pouch tissue, increased tumor growth . LLLT/PBM at a dose of 150 J/cm2 appeared safe, with only minor effects on B16F10 melanoma cell proliferation in vitro, and had no significant effect on tumor growth in vivo. Only a high power density (2.5 W/cm2) combined with a very high dose of 1050 J/cm2 could induce melanoma tumor growth in vivo . In a mouse model to study LLLT/PBM effects on UV-induced skin tumors, the experimental mice received full body 670 nm LLLT/PBM delivered twice a day at 5 J/cm2 for 37 days, whereas controls received sham LLLT/PBM . No enhanced tumor growth was observed, whereas there was a small but significant reduction in tumor area in the LLLT/PBM group, potentially related to a local photodynamic effect or LLLT/PBM-induced antitumor immune activity.
The results from these studies suggest that different tumor cells have distinct responses to specific LLLT/PBM parameters and doses. In part, these differences may be also explained by variations in the cellular microenvironment, since these have been shown to affect cellular signal transduction pathways to LLLT/PBM exposure . The microenvironment of tumor cells varies among in vitro studies and differs significantly from that found in animal models. Moreover, this difference implies that the potential of LLLT/PBM to enhance proliferation of tumor cells in vitro does not necessarily translate into harmful effects of LLLT/PBM in cancer patients.
A recent RCT in which LLLT/PBM was administered for prevention of OM during CRT in HNC patients (diagnosed with SCC of the nasopharynx, oropharynx or hypopharynx), reported that at a median follow-up of 18 months (range 10-48 months), patients treated with LLLT/PBM had better locoregional disease control and improved progression-free or overall survival .
Current evidence suggests that LLLT/PBM in the red or NIR spectrum, with an energy density of 1-6 J/cm2 is safe and effective, which suggests that it should not be withheld from HNC patients. However, the potential effect on dysplastic and malignant cells has not been definitively resolved. Virtually all studies have focused on cell-based assays rather than conventional xenograft or orthotopic animal models. And the results of in-vitro investigations has been largely dependent on the experimental design and selection of target cells. Key biologic effects of wavelength, energy density and time/duration of exposure are important measures of LLLT/BPM characteristics that must be recognized and evaluated. As robust evidence for the lack of malignant cell protection or enhancement of tumor growth has not been published, vigilance remains warranted.