As expected IMRT caused a significant and detrimental effect on salivary glands; even after 6 months reducing SFR and TP to a third of pre-IMRT levels, but both recovered to some degree by 12 months. Significantly there was no effect of IMRT on salivary mucin or IgA levels at either timepoint. These proteins are crucial for the lubricating and immunological defence of the oral mucosa by binding to the oral epithelial cells as the pellicle [14]. Although no saliva sample was collected either during or immediately after IMRT, it is assumed that this pattern of salivary inhibition would be similar or worse during IMRT. Nearly all patients receiving IMRT experienced some degree of OM during IMRT and some still had OM at 6 months. This suggests OM caused by IMRT permanency is not connected by a loss of the lubricating components of saliva (mucins) but instead may relate to other factors such as direct effect of RT on epithelial cells, altered oral microbiome or alterations to the oral mucosa binding proteins (cell membrane-bound proteins).
Undoubtedly damage occurred to the main secretory apparatus (serous acinar cells) of the major salivary glands since markers for parotid acinar cells (amylase) and submandibular serous acinar cells (cystatin) were both greatly reduced at 6-months post-IMRT as well as the salivary flow which is a product of the acinar cells [25]. However, this reduction was temporary and both glands had recovered to some extent by 12 months. In agreement with other reports, parotid gland appeared to recover faster since amylase rate had returned to pre-IMRT levels after 12 months whereas cystatin levels had not. Typically resting UWMS is composed of relatively more submandibular/sublingual saliva compared with parotid saliva [19–26]. Despite acinar cell damage to both major glands, there were signs that other parts of the glands were not affected. For example, mucin, which comes from the mucous acinar cells of the submandibular and sublingual glands, and IgA secretion rates were not decreased by IMRT. Dimeric IgA translocation occurs by pIgR expressed by both acinar and ductal cells [27]. There was a trend of increased IgA secretion at T2 similar to other studies [23–28–29]. This may reflect increased plasma cell infiltration into saliva related to damage although the salivary IgA secretion rate is typically limited by the parenchymal translocation of pIgR and not the supply of dimeric IgA by plasma cells [27]. Although an increased mucin concentration in a reduced salivary volume might negatively affect the rheological properties of saliva, increasing viscosity, that would not impair the ability to lubricate the oral cavity. Therefore, it would be interesting to investigate other factors which could affect oral lubrication. The most obvious would-be MUC1 the oral epithelial cell membrane-bound mucin that anchors salivary mucins to the surface [30]. In addition, altered glycan composition could change mucins configuration, resulting into tight packed globular aggregates with a reduced capacity of retaining water [13–31].
Albumin secretion rate was unchanged at T1 and surprisingly showed a significant decrease at T2 compared to T0. This suggests that the oral side effects of IMRT, particularly inflammation, did not affect the transfer of albumin into saliva in contrast to other reports [32]. These outcomes are in contrast with other studies, albeit ones which studied enamel, mucosal pellicle rather than saliva, which observed increases in albumin concentrations in 9 HNC patients’ post-RT (3-4 months) in comparison with pre-RT measurements, even though all patients underwent laser therapy to treat OM [33].
Regarding primary tumour site, most patients presented locations that were anatomically close to each other and nearby parotid and submandibular glands. Therefore, it is possible that these two salivary glands were affected by the toxic side effects of IMRT [19].
There was a negative and significant association between IgA concentration with OM occurrence and maximum severity during the cancer treatment. This indicated that patients with lower pre-IMRT IgA saliva concentrations could be more prone to develop grade 2&3 OM (Grade 2&3 mean IgA concentration 10.4, Grade 1 mean IgA concentration 17.7). This should be explored during the IMRT, also baseline records with non HNC patients in order to explore more deeply the nature of this association.
It has been established in the literature that an altered concentration of IgA at mucosal surfaces will impair host microbial homeostasis, adherence, and protection from bacterial infection, altering bacterial diversity and biofilm formation[17–34] . Moreover, IgA is vital for commensal bacterial colonisation in gut mucosal tissues [34] and may bind certain bacteria facilitating colonisation of the oral mucosa [35] to maintain a healthy and functional mucosal barrier which is critical during OM onset/development.
Thirty-seven patients (94%) developed OM during RT, with no patients presenting severity grade 4 however, 30% of the patients suffered OM severity grade 3. These findings are similar to Orlandi et al. (2018) [36] who reported severe acute OM (G≥3) in 30% of HNC patients treated with IMRT. Equally, to Oba et al 2021 [37] who reported 0% of grade 4 cases in HNC patients that underwent IMRT.
Additionally, it was determined that tumour location was not a factor in OM as there was no difference amongst different tumour locations, nor unilateral or bilateral radiation dose and tumour stage regarding OM onset or severity.
Significant associations at T1 and T2 between OM and reduced SFR were revealed in all subjects. This association alludes to the influence of the oral environment, especially the role of saliva throughout the development of OM/severity with regards to mucosal wetting, lubrication and bacterial protective functions and colonisation [38–39]. However, the clinical relevance of such findings is dependent on the changes occurring within the composition of saliva [39]. There were significant positive associations when measuring TPSR, as well as concentrations, with OM at T1. This is of particular note as findings revealed that TPSR was significantly reduced post-radiotherapy, thus altering saliva properties.
The significant associations between the number of patients presenting persistent OM (n= 6) and mucin5B and 7 secretion rates at T1, were similar to a previous report developed by Jham et al., (2008) [40].
Onset of OM caused by the direct effect of IMRT, also can be affected by patient-related factors, these results at T1 may indicate that the protective mucosal layer was changing and no longer helping to maintain mucosal integrity during RT, when the OM cases reached 37 in total, with 11 grade 3 severity patients. Furthermore, the reduced SFR along with an increased mucin5B and 7, probably affected the saliva’s viscoelasticity, making saliva “stickier” and more viscous so reducing its functional value, resulting in an impaired protective barrier [41].
In summary, analysis suggest that IMRT can limit the damage to salivary glands and importantly permit the recovery of function. This study provides an insight into the implications of loss of salivary lubricating factors as a patient related factors associated to OM. Although altered protein composition and a reduced salivary volume will impair several functions including swallowing, speaking, communication, oral discomfort, taste acuity, oral homeostasis, maintenance of the symbiotic microorganism balance in the oral cavity, as well as a reduced mucosal moistening and protection increasing the risk for ulcers during their cancer treatment[15] . Furthermore, the clinical importance of this study becomes clear when considering the associations between clinical and biochemical data in order to identify potential markers for increased susceptibility to severe OM, in a larger group of HNC patients. To date, there are no therapeutic agents to resolve or reduce the duration of OM also remains a dose-limiting factor in HNC treatment. Patients affected by OM tend to interrupt or even terminate their treatment course early, because they undergo significant deterioration in their health related QoL, despite adversely affecting the survival rate outcomes ([6–12–16–27–42] 53).
A limitation in OM research is the lack of universal, objective, and standardised indices for data collection. Mucosal evaluation outcomes depend mainly on the consulting physician’s clinical experience and training. There is little consensus among researchers regarding these evaluation indices[7–43] since there is a lack of evidence of any superiority among these scales. Nevertheless, the WHO toxicity scale has been used extensively in clinical OM research since 1979 [43]. The number of medical appointments, examinations, and tests before the commencement of IMRT, along with several side effects that appeared after cancer treatment, can make sample collection challenging, so reducing study protocol compliance. However, the dropout rate in this study was low (7 patients). The reduced salivary flow rate after IMRT makes difficult to collect enough amount of sample for biochemical analysis, thus everything must be accurately designed and planed, to avoid variation. Another limitation of this study was the sample size; therefore, conclusions should be treated with caution. Larger longitudinal studies are needed to confirm the outcomes. Additionally, there was no sample collection during cancer treatment, which could have provided information during OM onset, thus it would be needed a prospective study including sample collection during IMRT to assess temporal aspecs of OM onset and severity. Finally, the heterogenicity of tumour sites could impact in the lower rate of severe grade 4 of OM cases.