108 Methods Patients were included by convenience sampling when they were 18 years or older, were diagnosed with oral, oropharyngeal, hypopharyngeal, or laryngeal cancer, and were treated with a curative intent at the University Medical Center Utrecht (UMCU), the Netherlands, between September 2014 and June 2018. Patients with recurrent or residual disease, cognitive impairments, and patients having trouble understanding or reading the Dutch language were excluded. All patients signed written informed consent before participation. The study protocol of this prospective cohort study was approved by the Medical Ethics Committee of the Netherlands (NL45051.029.13), and is part of the NET-QUBIC research.10 Patient data about age, sex, tumor stage,11 tumor site, and treatment were collected. Patients were assessed before primary treatment (baseline, M0), and 3 (M3), 6 (M6), 12 (M12), and 24 months after treatment (M24). At every assessment, the Mixing Ability Test measuring masticatory performance was performed. Mixing Ability Test The Mixing Ability Test (MAT) consists of two layers of wax, with the colors red and blue (Plasticine modelling wax, non-toxic DIN EN-71, art. nos. crimson 52801 and blue 52809, Stockmar, Kalten Kirchen, Germany).3,12-14 The total thickness is 3 mm, with a diameter of 30 mm. The outcome variable is called the Mixing Ability Index (MAI), and ranges between 5-30, where a lower MAI score implies a better mixed tablet and better masticatory performance. A subject was asked to chew on this tablet 20 times in order to mix the two colors. The tablet was then flattened, pressed to a thickness of 2 mm, and scanned on both sides using a high-quality scanner (Epson® V750, Long Beach, CA, USA). The scanned images were processed using Adobe Photoshop CS3 extended (Adobe, San Jose, CA, USA). The histograms of both sides of the flattened and scanned wax tablet were added to obtain red and blue intensity distributions. The spread of the color intensities was measured, and a mixing ability score was calculated.13 In previous research, this test showed a good reliability (ICC=0.886) when comparing test and retest.15 To identify patients with masticatory dysfunction, a cut-off value was calculated, based on a value larger than 2 standard deviations from the mean value of healthy subjects, as calculated in previous research. A cut-off value of ≥20.5 indicated masticatory dysfunction.15 Statistical Analyses Descriptive statistics were used to describe the study population. A Kruskal-Wallis H test was performed to examine differences in age between different tumor sites, and a chisquare test was run to test for differences in sex, primary treatment, and tumor stage between tumor sites. A linear mixed-effects model (LMM) with the MAT as dependent
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