Relationship between nutritional biomarkers and occlusal status in gastric cancer patients using the Eichner Index: A cross sectional study

Abstract

Gastric cancer is one of the most lethal forms of cancer worldwide, with an estimated 951,600 new cases and 723,100 deaths in 2012 [1,2].Despite early detection, improvements in surgical procedures, and availability of advanced chemotherapeutic options, long-term survival is still unsatisfactory due to the associated local recurrence or distant metastasis.The immune and nutritional status of patients have been reported to be associated with postoperative outcomes in malignant tumors [3][4][5][6].The nutritional status and systemic in ammatory responses in patients with cancer are considered to be useful prognostic factors [7][8][9].
The nutritional status of patients with cancer is often evaluated by physical examination and blood biochemical ndings such as albumin [10].Patients with gastric cancer who are immunocompromised due to preoperative undernutrition or systemic in ammatory responses are often have delayed wound healing, develop complications, and have increased mortality rates [11][12][13].Malnutrition often causes changes that can lead to a poor prognosis [14,15].Therefore, it is important to develop a precise biomaker to objectively evaluate patients with malnutrition.Various biomarkers that re ect the in ammatory state, nutritional status, and immunocompetence of patients have been reported as valuable prognostic factors [16][17][18][19][20].One method of evaluation is to measure plasma components such as C-reactive proteins (CRP) or serum albumin (ALB) levels, another involves measuring blood cells such as neutrophil or lymphocytes levels, and a third method involves the combination of these two.Use of a nutritional evaluation index has advantages and when used with other nutritional indexes, it can help to improve the diagnoses of patients.Due to the limitations of using a single index, we chose appropriate nutritional indexes depending on the disease stage and the type of therapy patients received [16].
Preoperative undernutrition is due to multiple biological, physiological, and pathological factors, one of which is a decreased oral function.Oral dysfunction, which causes undernutrition, includes impaired masticatory ability due to tooth loss, dry oral mucosa, and infection.Such conditions affect food choices and result in a reduced quality of life (QOL), development of severe complications, and increased mortality rates [16][17][18][19][20].If cancer progresses, the nutritional status of patients gradually worsens.
However, patients with a decreased oral cavity function with no disease progression tend to tilt towards being malnourished.Hence, a decrease in the oral cavity function results in a poor preoperative nutritional status during gastric cancer surgery, with the possibility of an increase in the occurrence of postoperative complications.Although there have been studies that investigated how a decrease in the number of teeth and an impaired masticatory function affect nutritional status, studies that determine the association between nutritional status before gastrointestinal cancer surgery and oral conditions of patients are few.
Speci cally, no study has focused on the relationship between biomarkers and occlusal zone support status exhaustively.In a previous study, we reported that the Prognostic Nutrition Index (PNI) differed signi cantly between group A (those with occlusal zone support) and group C (those with no occlusal zone support based on the Eichner classi cation [21][22][23].There are two kinds of biomarkers for evaluating systemic in ammation and nutritional status.Currently, it is uncertain whether the intraoral state of the patient affects either blood cells or plasma components and to determine these effects would require a novel evaluation approach.We showed in our previous work that the assessment of nutritional status using the PNI and ALB levels is important in preoperative dental evaluation.
In the present cross-sectional study, we investigated whether some biomarkers (Glasgow prognostic score [GPS], neutrophil-lymphocyte ratio [NLR], controlling nutritional status [CONUT], and PNI), preoperative nutritional markers in patients with gastric cancer, would differ according to occlusal support zone status.
A change in food selection is often observed with a decline in masticatory e ciency due to tooth loss and malocclusion [24].People with chewing di culties eventually consume less nutritious foods such as vegetables, fruits, meats and grains [24][25][26].As a result, blood levels of vitamins and protein are reduced; however, the nutritional evaluation index is thought to be more accurately re ected by evaluating plasma protein levels [27,28].This is because a change in diet is considered to have a bigger impact on plasma proteins than on other hematopoietic mechanisms in the short term.For this study, using the GPS, CONUT, and PNI, we evaluated the plasma components that are affected by occlusion support zone status.Additionally, we used the NLR to assess how other blood components are affected by occlusion support zone status.From this assessment, we determined how the occlusion support zone status is associated with various biomarkers in the blood.

Subjects
This cross-sectional study enrolled 145 patients who were pathologically diagnosed with primary gastric cancer, and underwent a dental examination before surgery at the Nagoya Ekisaikai Hospital between September 2014 and March 2019.Of these patients, 31 who had recurrent cancer, metabolic diseases (e.g., diabetes mellitus), or incomplete data were excluded.The present study, 114 patients (85 men and 29 women) were included.

Assessment of gastric cancer and general conditions
Gastric cancer was diagnosed by consensus among skilled physicians.While assessing gastric cancer, the onset site and cancer stage was determined based on the results of endoscopy, computed tomography, and magnetic resonance imaging performed at the department of gastrointestinal surgery.The body mass index (BMI) of each patient was also calculated.Blood samples were collected on admission, during the presurgical examination (within one month before surgery).From these blood samples, ALB levels, total lymphocyte counts, and CRP levels were measured.Based on these preoperative blood test results, the severity of in ammation was assessed.

Evaluation of nutritional biomarker
We evaluated four kinds of nutritional biomarkers.The biomarkers evaluated were GPS [29,30], NLR [31,32], CONUT [33,34], and PNI [17,18] as described in Table 1.The GPS was calculated using preoperative CRP and ALB values.Patients with raised concentrations of CRP (over 10 mg/L) and hypoalbuminemia (ALB < 35 g/L) were given a score of 2. Those with raised concentrations of CRP without hypoalbuminemia were given a score of 1. Patients who did not have raised concentrations of CRP were given a score of 0 regardless of their ALB status.Neutrophil-lymphocyte ratios were also calculated.A neutrophil-lymphocyte ratio of ≥ 5 was given a score of 1; if it was < 5, the score was 0. PNI was calculated on the basis of serum ALB levels and peripheral blood lymphocyte count using the following formula.The serum albumin concentration, total peripheral lymphocyte count and total cholesterol concentration were used to obtain the CONUT score.The scores were added up and patients were categorized into the following groups based on the cumulative score: normal (0-1) mild disorder (2-4), moderate disability (5-8), and severe disability (> 8).

Assessment of oral conditions
Oral conditions were assessed by two dentists who were blinded to patients' nutritional status in the present study.In the oral examination, the remaining teeth excluding residual dental roots were counted, and the occlusal support zone status was assessed according to the Eichner classi cation [7].The Eichner classi cation is a method used to evaluate occlusion support zone status based on the remaining teeth.The zone is de ned as a site supporting occlusion.In a healthy dentition composed of opposing tooth pairs in the right and left premolar and molar regions, occlusion is supported by 4 occlusal supporting zones.The Eichner index categorizes the occlusal status into three main groups (A, B, and C).The number of remaining teeth and Eichner classi cation are so objective that they can be regarded as very reliable.
In patients using dentures, their dentures were examined by a dentist.The occlusal supporting zones were assessed for contact between opposite teeth pairs.

Statistical analysis
The number of cases in the area during the study period determined the sample size.We also examined whether the values of the four biomarkers differed among the 3 Eichner Classes A, B, and C. We considered whether there was a difference in GPS and CONUT frequencies between the three Eichiner groups.Categorical data (GPS and CONUT) were analyzed using χ 2 tests, except when expected cells were found to be less than 5, in which case, Fisher's exact test was employed.The Shapiro-Wilk test was used to assess the distribution of preoperative NLR and PNI values, and the normality was con rmed at a 5% signi cance level.Because NLR and PNI were not normally distributed (P = 0.000003493 and 0.02542), the three groups were compared using the Kruskal-Wallis test, which is a non-parametric test.In addition, the Steel-Dwass multiple comparison tests were performed to determine which of the three groups showed differences in NLR and PNI.All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).More precisely, it is a modi ed version of R commander designed to add statistical functions frequently used in biostatistics [24].Based on the effect size observed in our study, the estimated sample size was 44 patients per group and a total of 88 patients.Sample size estimates were based on the following: standard deviation within groups 5.0, αlevel, 0.5 and power of 80%

Patient characteristics
None of the markers of systemic and oral conditions were normally distributed.Patients were classi ed into three groups according to the occlusal support zone status based on the Eichner classi cation to compare each marker.

Oral conditions
The mean number of remaining teeth was 18.1 ± 9.5 teeth (range, 0-31 teeth).Even among patients with the same number of remaining teeth, if the number of occlusal supporting zones differs, their nutritional status may be differently affected.Thus, differences in nutritional biomarkers were analyzed according to the Eichner classi cation.The nutritional biomarkers are shown in the Table 2

according to Eichner
Classes A, B, and C. The CRP levels were 0.5 ± 1.1, 0.5 ± 0.7, and 1.7 ± 3.3 in groups A, B, and C, respectively (P = 0.01), indicating signi cant differences among the three groups.The ALB levels were 3.9 ± 0.45, 3.68 ± 0.5, and 3.43 ± 0.7 in groups A, B, and C, respectively (P = 0.002), indicating signi cant differences among the three groups.(Table 2) The GPS is a common score used to assess levels of in ammation.The score assessments revealed frequencies of 32 GPS level 0, 4 GPS level 1, 5 GPS level 2, and 4 GPS level 3 in group A, 21 GPS level 0, 9 GPS level 1, 6 GPS level 2, and 6 GPS level 3 in group B, and 11 GPS level 0, 2 GPS level 1, 3 GPS level 2, and 11 GPS level 3 in group C (P = 0.0179), indicating signi cant differences among the three groups.In addition, multiple comparisons revealed no difference in GPS levels between groups A and B, and between groups B and C (P = 0.615, P = 0.23), but signi cant differences were observed between groups A and C (P = 0.033) (Mann-Whitney U test) (Fig. 1).
The PNI is calculated based on the serum albumin concentration and peripheral blood lymphocyte count.
The values for patients in this study were 42.6 ± 4.9, 40.3 ± 5.8, and 37.1 ± 7.6 in groups A, B, and C, respectively (P = 0.0048), indicating signi cant differences among the three groups.In addition, multiple comparisons revealed no difference in either ALB levels or PNI between groups A and B, and between groups B and C (P = 0.29, P = 0.06), but signi cant differences were observed between groups A and C (P = 0.00097) (Mann-Whitney U test).These results revealed that PNI values were lower in Group C with poor occlusal support than in Group A with stable occlusal support (Fig. 3).
The CONUT score is calculated according to serum albumin concentration, total peripheral lymphocyte count and total cholesterol concentration.The score frequencies for this assessment were 25  CONUT level 3, and 4 CONUT level 4 in group C (P = 0.03), indicating signi cant differences among the three groups.In addition, multiple comparisons revealed no difference in GPS levels between groups A and B, and between groups B and C (P = 0.39, P = 0.6), but signi cant differences were observed between groups A and C (P = 0.04) (Mann-Whitney U test) (Fig. 4).
Thus, the present study revealed that the GPS, PNI, and CONUT were affected by the numbers of remaining teeth and occlusal supporting zones.

Discussion
The oral cavity is an important organ for nutrient intake, and nutritional status is affected by impaired oral function due to hypofunction of the temporomandibular joints, masticatory muscles, missing teeth, and impaired swallowing function [35][36][37].In the present study, the examination of occlusal support zone status and nutritional biomarkers revealed signi cant differences in GPS and CONUT levels and PNI values.The Eichner C group without occlusal support had higher GPS levels than did Group A, and the CONUT levels revealed moderate or severe malnutrition; PNI values were low in this group.However, there was no signi cant difference in NLR between the three groups.Furthermore, nutritional evaluations using the protein levels of the blood revealed signi cant differences in the oral cavity function failure among the three groups; whereas, a signi cant difference was not found in nutritional evaluation using the blood cells.Therefore, the change in diet due to oral cavity function disorder reduced plasma proteins such as albumin and cholesterol, and was thought to result in undernutrition and poor immunocompetence.The results suggested that impaired oral function due to oral diseases and tooth loss reduces dietary intake and is associated with undernutrition.The correlation between the number of residual teeth and nutritional status has been reported in many studies, and the results of the present study are comparable with those of such studies.Meanwhile, the bite force varies depending on periodontal disease and stability of the remaining teeth, and it affects the food choice and nutritional status.Thus, even among patients with the same number of remaining teeth, their conditions may differ depending on the sites and arrangement of the remaining teeth.
Plasma proteins decrease due to undernutrition arising from poor caloric intake.The decrease in plasma proteins reduces immunocompetence, and the presence of in ammation worsens the situation.Although the Eichner classi cation is based on the assessment of the occlusal support zone status at the time of clinical examination, it may help make a diagnosis of oral frailty [37] after a certain period because the simultaneous loss of multiple teeth rarely occurs.Thus, this classi cation was used in the present study.
A comparison among the 3 Eichner groups revealed signi cant differences in GPS, CONUT, and PNI among the groups, and multiple comparisons revealed a signi cant difference between groups A and C. It is rare to lose many teeth in a short period of time; the patients in group C might were likely undernourished before they were diagnosed with gastric cancer.
In patients with poor preoperative nutritional status, the relative risks of postoperative complications and in-hospital death are considered to be increased by 2-4 folds.Cell-mediated immunity is most affected by undernutrition.Undernourished people exhibit atrophy of the thymus, reduced peripheral T cell counts, reduced CD4/CD8 ratios (CD4 dominant), and atrophy of lymph nodes, spleen, and gut-associated lymphoid tissue (GALT), which is the immune tissue of the intestinal tract.Atrophy of GALT reduces the immunity of the intestinal mucosa and causes gastrointestinal infection.In addition, severe malnutrition reduces immunoglobulin levels.On the other hand, neither neutrophil nor peripheral B lymphocyte count decreases; but the phagocytic capacity, bactericidal capacity, and complement production are reduced [38][39][40][41].
Furthermore, systemic in ammatory responses are reported to re ect immune responses and are associated with prognosis.Systemic in ammatory responses are useful for predicting prognosis via a mechanism different from that of conventional tumor markers [41,42].Many tumor markers are tumor antigens and substances secreted by tumors that are measured directly and assessed.On the other hand, systemic in ammatory responses are an indirect measure of cancer progression.This is considered to be due to the following mechanism: host immunity is activated by tumor recognition, and proin ammatory cytokines are released in blood as intracellular mediators.Consequently, acute in ammatory proteins increase; thus, host immune responses are enhanced as tumors progress [43].
Biomarkers for assessing systemic in ammatory responses and the nutritional status include the Glasgow prognostic score [29,30], neutrophil/lymphocyte ratio [31,32], lymphocyte/monocyte ratio [44,45], and CRP/ALB ratio [46].These markers are calculated based on serum protein levels (e.g., CRP and ALB), cell counts (e.g., neutrophil, lymphocyte, and monocyte), and ratios of these values.Each nutrient rating system has its intrinsic characteristics.There are limitations to the use of a single index; thus, we chose appropriate nutritional indexes and evaluated them based on the disease stage and the therapeutic option employed.
The GPS was the index reported in non-small lung cell cancer by Macmillan, and the GPS combines CRP and ALB and evaluates blood protein moieties primarily.The GPS has been reported in various cancer studies to be a prognostic marker.The thing that comes to have a poor prognosis is reported in the systemic in ammatory reaction sthenia case like GPS Category 2, 3.The poor case shows systemic in ammatory such as the GPS Category 2 and 3 and undernutrition.The CRP level of patients with cancer re ects IL-6 levels in the blood, and a persistent increase in IL-6 levels suggests in ammation of the carcinoma tissue [47].Whereas, the progression of periodontal disease and the presence of dental caries cause odontogenic infectious diseases and an increase in CRP.
However, between the three Eichner groups, a signi cant difference was not found in CRP levels.The effect on GPS had little impact on teeth and chronic odontogenic infectious diseases in this examination, while for effect on GPS category, ALB was thought to be more important.Systemic in ammatory responses are assessed as changes in biochemical data that result from activation of the immune system by the presence of tumors and secretion of IL-6 and other proin ammatory cytokines [47][48][49].
NLR is a nutrient rating system using blood cells (neutrophils and lymphocytes).Lymphocytes have been used as one of the nutritional evaluation indexes for a long time [50].We are used for an index of the immunocompetence that the lymphocytes are related to tumor immunity and act for a tumor restrainingly.The lymphocytes are related to tumor immunity and act for a tumor restrainingly [51,52].
Whereas neutrophils increase due to in ammation and induce the production of chemokines and cytokine, the produced chemokines and cytokines enhance the growth, invasion, and neovascularization of tumors.Therefore, tumor growth is closely associated with in ammation [53,54].NLR, which evaluates CRP and ALB ratios, is reported as an oncological prognostic marker similar to GPS.A signi cant difference was not found in this examination between the three Eichner groups.NLR is considered to mirror a balance between the innate and adaptive immune mechanisms [54].The NLR can re ect the initial innate immune mechanisms (involving cells such as neutrophils and macrophages that provide a nonspeci c response), which triggers the adaptive immune mechanisms (T-cell/B-cell mediated, and partly PLT stimulated) that result in periodontal destruction.In cases of high NLR values, periodontitis becomes more severe [52].It is reported that NLR is useful for clinical evaluations of periodontitis.A mouth cleaning state is good in Eichiner A where many residual teeth are present in, and in ammatory reaction is poor.Additionally, there is no effect as we raise CRP because Eichner C has few residual teeth causing odontogenic infectious diseases.The difference was not found in such situation.
Whereas there are many patients that a lot of residual teeth have good mouth cleaning state in Eichner A. Also, Eichner C has few residual teeth and does not reach it before we greatly change CRP even if periodontal disease is severe because there are few teeth causing in ammation.A difference was not found from such situation.PNI and CONUT are indexes that are dependent on the evaluation of lymphocyte counts, ALB, and cholesterol.It has been reported that it has a poor prognosis in low level of PNI and the extensive malnutrition category of CONUT.The lymphocytes have antitumor immunity eligibility.The lymphocytes commit a tumor restrainingly and have antitumor immunocompetence [53].
Cholesterol half-life in the blood is eight days, shorter than 21 days for ALB, and re ects malnutrition subtly [24][25][26]55].Signi cant differences were found between the three groups and PNI and CONUT proved to the more re ective of undernutrition in group C than in group A. Wakai et al. [55] reported that as the number of teeth decreases, people consume fewer vegetables but their total calorie intake increases with an increase in the consumption of carbohydrate such as rice, and other confectionaries.If the occlusion status is poor, carbohydrates and glucide play key dietary roles.Simultaneously, there is a reduced intake of neutral fat or proteins.As a result, these indexes are thought to worsen.
The present study has several limitations.Nutritional status is affected by many factors.In addition to oral ndings such as prosthodontic treatment with dentures and conditions of the remaining teeth, other factors including socioeconomic strata, income levels, and acquired behavioral patterns are also very important [52].It was impossible to control all of these potential variables in the present study.Furthermore, to determine whether dentures functioned well, factors that can in uence outcomes such as the levels of technical skills of dentists, designs of dentures, the di culty of cases, skills of dental technicians who fabricate dentures, perfection levels of dentures, and the actual use of dentures, should be assessed.However, there are no standardized methods to assess these factors, and only a few studies have reported the evaluation of these factors.These factors should be assessed in future studies.
Because the present study is a cross-sectional study, no causal relationship could be determined.Thus, we cannot state that the number of teeth or occlusal status directly affects nutritional status.
An early transition to oral intake is essential in the treatment of gastric cancer.The reasons for this are not only that oral intake is the best enteral nutrition, but also that eating through the mouth is an important activity, which is associated with human dignity and affects QOL.In addition to the problems of undernutrition, prolonged lack of oral intake leads to a disuse hypofunction of the eating and swallowing muscles.Thus, a delayed start of oral intake due to complications, such as postoperative ruptured suture, should be avoided, and efforts should be made to precisely diagnose and improve preoperative undernutrition.Given the importance of the function of the remaining teeth for nutrient intake and the unduly signi cant effects of impaired masticatory function on nutritional status, dentists and dental hygienists in a nutrition support team play an important role in maintaining and improving oral function, which is important for nutrient intake.For example, the oral cavity should be kept clean, and the periodontal condition should be maintained.In addition, patients with occlusal disharmony may be undernourished, as an underlying condition, even if they are satis ed with denture use.Thus, occlusal disharmony is suggested to be a possible factor for the consideration of preoperative nutritional supplementation.Based on this study, there are two conclusions that can be drawn regarding malnutrition and in ammation in patients with gastric cancer patients with oral health conditions: 1) There are direct effects of occlusal abnormalities on dietary intake, and 2) There is indirect action of systemic in ammatory conditions such as odontogenic infectious diseases on acute protein synthesis.
Further studies on oral health and its role in malnutrition and in ammation are warranted.A long-term prospective study and intervention study are necessary to completely evaluate the association among systemic in ammation in oral health, malnutrition, and gastric cancer.

Conclusions
The association between the nutritional biomarkers and dental conditions of patients with gastric cancer with different occlusal statuses was examined.Decreases in the occlusal supporting zones were correlated with undernutrition.Patients with fewer occlusal supporting zones had lower ALB levels and PNI, and GPS and CONUT levels were found to be reduced in undernourished patients.The Eichner C group was inferior to other groups in GPS, PNI, CONUT.The occlusion status affects humoral ingredients such as albumin.This study emphasizes an important role of oral health in improving malnutrition in gastric cancer.Furthermore, it shows that it predicts the decrease of a nutrient in the gastric cancer and the caloric intake that the oral cavity appearing by the loss of teeth is in bad health.Furthermore, oral functional decline due to the tooth missing shows that we predict a decrease in nutrition of patients with gastric cancer and systemic in ammatory reaction.