Patient case: 26-year-old female with chondrosarcoma; genetic feature: IDH1

Clinical trial: The Characterisation of RT Caries.

Summary: Head and neck cancer represent 4-5% of all of cancer diagnosed in the UK, the first choice of treatment it is Radiotherapy (RT). RT is known to produce a wide range of oral side effects such as hypo-salivation, caries, xerostomia, mucositis and osteoradionecrosis.
  Hypo-salivation may lead to a shift in diversity of the bacterial population altering oral homeostasis. RT may also cause a variation in saliva protein composition which can affect its rheological properties and host defence increasing the possibility of caries development. Additionally, the effects of RT on the organic and inorganic structure/ composition of teeth and the possible relation with caries risk have not been investigated so far.
  Mucositis is another frequent oral complication of RT and it is estimated that more than 80% of the treated patients will develop this condition.
  Longitudinal studies assessing all these conditions before and after cancer treatment have not been reported in literature to date.
  Therefore, the main objective of the proposed study is to assess the chemical composition of the dentine matrix, the variations in salivary proteins and in oral microbiome occurring in Head & Neck cancer patients undergoing RT. These data will be collected and a complete oral assessment with 18 month follow up will be performed. All these data will be computed to demonstrate the associations between RT and caries risk to demonstrate possible mechanistic causes.

Detailed description: 1. Introduction
  Head and neck cancer is the sixth most common cancer in the world representing 4-5% of all malignancies . Head and neck cancer (includes oral, nasal cavity, salivary glands, paranasal sinus, pharynx, and larynx) is one of the top 10 most diagnosed cancers in the UK . According to Cancer Research UK, there were 7591 newly diagnosed with oral cancer in 2013 in the country, representing the 14th most common cancer in the UK.
  Radiotherapy (RT) is the first choice treatment for the majority head and neck cancer patients, which is a localized treatment to control the tumour . More than 50% of head and neck cancer patients receive RT delivered in fractions of 2 Grays (Gy) daily during 5 days per week until the end of their therapy dosage (mean cumulative dose 55-77 Gy). RT stimulates cellular changes that destroy the tumor cells, but may affect dramatically the healthy oral tissues altering the homeostasis of this complex environment. RT is the cause of a wide range of adverse oral tissue reactions such as permanent damage of the major and minor salivary glands (hyposalivation, xerostomia), modification of connective and vascular tissues and a reduction the healing capacity especially in mucosal and mineralised tissues (mucositis, caries lesion, osteoradionecrosis). In order to try to diminish the adverse side effects of the RT three-dimensional (3D) computed tomography (CT) planned radiotherapy known as intensity-modulated radiotherapy (IMRT) has been developed .
  There are acute (mucositis) and late-onset adverse effects/sequelae that RT induces in healthy oral tissues . Radiation-induced dental caries is the most common late effect, but its origin and nature is not clear. RT related caries have a rapid onset as a result of a combination of factors such as shift in oral microbial community composition favouring acidophilic bacteria, hypo-salivation, poor oral hygiene and dietary alterations . The patients often have little/no pain, even in the most severe cases. Histological features show that the onset and development of caries is similar to non RT-related ones but the sites affected are different. Indeed RT-related caries is more frequent on the cervico-labial surface/ incisal edge of canines and incisors while non RT-related ones are more frequent in the interproximal/ occlusal areas of the posterior teeth (molar and premolars).
  Also, caries associated with RT has a rapid circumferential progression which may lead to tooth fracture. It is characterized by a localized lesion to the incisal or occlusal surfaces which diffuse and progress in relation to irregular and generalized erosion of the incisal edge of the tooth. In addition, mechanical changes (increased elastic modulus and stiffness) within the tooth structure increase the wear of the incisal and occlusal areas and could lead to complete fracture of the tooth.
  Tooth destruction is one of the most severe consequences of RT and it may produce complete edentulousness in 3 - 5 years. The dental management of RT patients is challenging. It is necessary to gather a multidisciplinary team (restorative dentists, periodontology, endodontic, dental hygienist, radiation oncologists, medical oncologists, and oral surgeons) to maintain oral health and improve quality of life (QoL). Almost 50% of cancer survivors reported depression, worry or tension related to oral dryness symptoms. Hence, RT sequelae detrimentally affect the quality of life of head and neck cancer patients.
  Overall, it is necessary the quantitative analysis of chemical matrix components, proteins and microbial changes that occur in patients' teeth/oral cavity, who have undergone RT, in order to determine the aetiology and pathophysiology of the two prevalent side effects (caries/mucositis) to correlate with preventive measurement and appropriate treatment care for this population. Finally, it is necessary to perform a complete oral assessment (dental/periodontal/xerostomia) using a validated index in order to create a baseline record before the treatment and a validated measured to compare after the RT and with healthy individuals.
  2. Aim Overall the aim of the proposed project is to perform a well-controlled and integrated longitudinal study of head and neck cancer patients by measuring variations in dentin matrix composition, salivary proteins, oral microbiome and clinical oral status upon RT to integrate them an finally to inform the dental community.
  2.1 Null Hypothesis There will be no differences in the chemical matrix components, proteins and microbial species in teeth/ oral cavity of head and cancer patients treated with radiotherapy compared with healthy subjects.
  There will be no differences in the chemical matrix components, proteins and microbial species in teeth/ oral cavity of head and cancer patients before and after the radiotherapy.
  3. Materials and Methods. 3.1 Characterisation of therapeutic radiation-induced dental caries study. Ethics application to Health Research Authority (IRAS Project ID:199100). All the samples of the study would be obtained from the Special Care dental Unit at KCL Dental Institute, Guy's Hospital and from the Dental Clinic at KCL Dental Institute.
  The collected samples will include saliva, biofilm, mucosal swabs, carious dentine and/or teeth. The samples collection will add approximately 10 to 20 min to the conventional treatment time. All the samples will be collected at 4-time points from the head and neck cancer patients during the 18 months follow up.
  Patients over the age of 16 in order to analyse permanent dentition and the age range for cancer patients are over 16 years old, both gender, without systemic diseases that can affect the salivary flow and host defence mechanism.
  Following these inclusion criteria the next patients are excluded younger than 16 (non-permanent dentition), unable to give the consent because they have to read and sign the consent, patients who have been administered antibiotics in the previous month because the oral microbiota could be altered promoting the opportunistic colonization, patients with systemic disease (such as Sjogren syndrome, depression, diabetes and high blood pressure) and under medication (antidepressants, including tricyclic compounds, benzodiazepines, antihypertensive: alpha-1 antagonists; alpha-2 agonists; beta blockers, which also alter salivary protein levels and diuretics) that could be alter the salivary flow and microbial composition.
  Sample size Calculation Statement The power calculation for this study was based on paired t-test for comparing the mean number of bacterial species found in Head & Neck cancer patients before and after treatment. A study with an effect size of 0.5 with 80% power to detect the true difference in a mean number of bacterial species before and after treatment, will require a total sample of 34 patients using the two-tailed test at 5% level of significance. The power calculation was carried out using Gpower 3.1.5.
  Proposed statistical analysis:
  Descriptive statistics will be used to summarize the sample characteristics and other clinical factors. Percentages and proportions will be used to present the incidence/ prevalence of caries and other clinical variables. The average number of bacterial species found in the Head & Neck cancer patients before and after treatment will be tested using paired t-test after checking for the normality assumptions. The normality assumption will be tested for all the parametric analysis using histogram and box plots. Repeated measures ANOVA will be used to test the variation of bacterial species within the oral cavity of Head and Neck Cancer patients who will undergo RT during 18 months follow up. The proportion of patients in different categories will be tested using Z test for testing proportions. The mean number of caries in the healthy controls and patients will be compared using independent t-test. Other relevant analytical methods will be employed to answer the research questions. The significance of the results will be assessed if p <0.05. All the analyses will be carried out using SPSS version 23.0.
  3.2.1 Whole Mouth Saliva sample Two Un-stimulated saliva samples will be collected by passive drooling method, following 30 min to 2 hour fasting and drink intake.
  The first sample will be collected by a passive drooling during 10 minutes.Donors will provide a 2-5 mL non-stimulated saliva by drooling into a 50 mL sterile conical polypropylene tube (Falcon; BD Biosciences, San Jose, CA, USA).
  In order to prevent the hyposalivation that head and neck cancer patients will present after the radiotherapy, a second sample will be collected from all of the patients recruited as it follows: each patient will rinse for 30 s with 10 ml of 0.9% normal saline solution and then spit into a 50 mL sterile conical polypropylene tube (Falcon; BD Biosciences, San Jose, CA, USA).
  3.2.2 Biofilm sample Supra-gingival dental biofilm sample will be collected from vestibular or lingual surface of the first molar with a sterile spoon excavator, each sample will be collected with a different spoon. No air-drying of the teeth would precede samples collection, thus to prevent biofilm disruption. The spoon will be moved over the enamel from mesial to the distal curvature of the tooth crown along the gingival margin and tooth surface border. Each sample will be released by agitation in Eppendorf tube containing Tris-EDTA buffer (Sigma-Aldrich, Irvine, UK).
  3.2.3. Carious dentine sample Carious dentine samples will be collected either from extracted teeth or directly from dentinal carious lesions of teeth that will be restored. After removing the superficial soft necrotic dentine layer with sterile spoon excavator (Ash, G5-Claudius Ash Ltd, Potters Bar, Herts, UK), the dentine carious sample will be collected at a level that represent the infected dentine of the lesion, evaluated clinically as a discoloured, sticky and soft to probe with a dental explorer. Dentinal caries will be collected with a sterile spoon excavator (Ash, G5-Claudius Ash Ltd, Potters Bar, Herts, UK), without local anesthesia, taking one scoop of caries. Cotton rolls will be placed buccally and lingually and suction will be provided to minimize saliva contamination during the process. Each sample will be transferred into an Eppendorf tube previously weighed, then the Eppendorf tube with caries inside will be weighed again. Finally, all samples will be suspended in 100 μL of nuclease-free water (Sigma-Aldrich, Irvine, UK).
  3.2.4. Mucosal swab Mucosal swabs will be collected using Sterile Catch-All™ Sample Collection Swabs (Epicentre Biotechnologies, Madison WI, USA) from the inside of the cheek by rolling the swab 20 times over the entire cheek with slight pressure applied. After the collection, the swab will be immersed in 750 μL of nuclease free water and the sponge would be pressed against the tube wall according to Manual of Procedures Human Microbiome Project Version 12.0.
  3.2.5 Teeth samples All teeth will be collected from the surgery clinic in KCL Dental Institute at Guy's Hospital.
  3.3 Sample storage All samples would be kept on ice and then stored at -80º C except the mucosal swabs samples that would be stored at -20ºC according to the followed protocol. All samples would be labeled and stored in the microbiology laboratory at Waterloo campus.
  Teeth will be identified, placed into purified water UHQ and refrigerated at 4ºC.
  3.4 Sample analysis Saliva samples would be processed for DNA extraction, using PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). Also, the protein content will be investigated usingPAS,BCA, AMYLASE KIT and ELISA analysis test.
  Mucosal swab protein content will be analysed using PAS and ELISA test respectively.
  DNA extraction will be performed in order to characterise the microbial diversity of the oral biofilm, whole mouth saliva, and caries of head and neck cancer patients before and after radiotherapy and compare with healthy control subjects using Illumina sequencing platform. This system will permit to determine the whole microbial species present in the oral environment, in order to determinate the most prevalent species in each subject in every location in the mouth (saliva, biofilm, caries and mucosa) to compare the differences and similarities. Additionally to analyse the species present in all subjects from each group and the variation of the whole microbiota after radiotherapy.
  In order to determinate the shift in saliva protein composition it will be analysed; total protein content and specific proteins concentration related to antimicrobial and remineralization salivary functions before and after radiation therapy in a longitudinal clinical study (18th months). On the other hand it will be compared the saliva protein composition between patients treated with RT and healthy subjects.
  Also, DNA will be extracted from the supra-gingival biofilm, mucosal swab, and dentinal caries samples.
  Using two photon microscopy will be possible to analyze the morphology and protein content of teeth collected from head and neck cancer patients before and after radiotherapy and compare with healthy subjects; two photon microscopy and Second harmonic generation (SHG) will be employed allowing the characterization of the collagen fibre structure and organization. Changes in the protein structure will reduce this SHG signal, providing the ability to detect a disruption in the structure of the collagen. Two-photon fluorescence microscopy has high resolution and allows the non-destructive optical sectioning of samples. Image analysis will be performed with ImageJ (Wayne Rasband, NIH, U.S.A.) in order to obtain information of collagen fibril size, diameter, spatial orientation and degree of organization.
  All samples would be analysed in the Biophotonics unit at Guy's Dental Institute.

Eligibility: Inclusion Criteria:
  Head and Neck Cancer Patients Male or female Over the age of 16. Received radiation treatment for Head and Neck cancer
  Control:
  1. Male or female
  2. Over the age of 16
  3. Good general health
  4. Permanent dentition at least 20 teeth
  Exclusion Criteria:
  Subjects younger than 16. Patients unable to give consent. Patients who have been administered antibiotics in the previous month.

Relevance to patient case: not relevant