Pathological examination of factors involved in PD-L1 expression in patients with oral tongue squamous cell carcinoma.

BACKGROUND: Tumor tissues comprise cancer cells and stromal cells, and their interactions form the cancer microenvironment. Therefore, treatments targeting cells other than cancer cells are also actively being developed, and among them, treatment targeting PD-1, an immune checkpoint molecule that is important in tumor immune evasion, has also been indicated for head and neck cancer. PD-L1, a ligand of PD-1, is expressed in both tumor cells and stromal cells, and the scoring system based on the combined positivity rates of both types of cells, the combined positive score (CPS), is used for predicting treatment effect. However, much is unknown regarding the expression of PD-L1. In this study, we histopathologically examined factors controlling the expression of PD-1/PD-L1. This study included 37 patients who underwent resection surgery for tongue squamous cell carcinoma in the Department of Oral and Maxillofacial Surgery at Tokyo Dental College Suidobashi Hospital. The expression levels of PD-L1, α-SMA, and p53 were assessed by immunohistochemical staining. RESULTS: Seven participants had CPS ≥ 20, twenty-four participants had 1 ≤ CPS < 20, and six participants had CPS < 1. The overall positivity rate of α-SMA, a marker for cancer-associated fibroblasts (CAFs), was 27% (10/37 participants), and the positivity rates of α-SMA for the three CPS groups were 85.7% (6/7 participants), 16.7% (4/24 participants), and 0% (0/6 participants), respectively. In addition, the overall positivity rate of p53 was 37.8% (14/37 participants), and the positivity rates of p53 for the three CPS groups were 71.4% (5/7 participants), 37.5% (9/24 participants), and 0% (0/6 participants), respectively. CONCLUSIONS: The expression of PD-L1 demonstrated an association with α-SMA and p53 positivity. In addition, compared with the expression of p53, the expression of α-SMA demonstrated a higher association with PD-L1 expression in patients with a high CPS. The abovementioned findings suggest that the interactions between CAFs, cancer cells, and immunocompetent cells may regulate the expression of PD-L1.

Impact of skeletal muscle mass on postoperative complications in oral cancer surgery.

BACKGROUND: Sarcopenia is characterized by a progressive and generalized loss of skeletal muscle mass and strength. The aim of this retrospective study was to investigate the impact of skeletal muscle mass on adverse events in free-flap reconstruction for defects after oral cancer resection. RESULTS: Of 120 patients, recipient-site adverse events occurred in 56 patients (46.7%), and recipient-site surgical site infections occurred in 45 patients (37.5%). Skeletal muscle index was significantly associated with recipient-site adverse events in univariate analysis (P < 0.05). Lower body mass index and skeletal muscle index were significantly associated with recipient-site surgical site infection in univariate analysis (P < 0.05). In the multiple logistic regression model, a lower skeletal muscle index was a significant risk factor for recipient-site adverse events and surgical site infections (adverse events odds ratio; 3.17/P = 0.04; surgical site infection odds ratio; 3.76/P = 0.02). CONCLUSIONS: The SMI at level Th12 was an independent factor for postoperative AEs, especially SSI, in OSCC patients with free-flap reconstruction.

Contrast-enhanced computed tomography of retromandibular vein position and course patterns relative to the mandible during sagittal split ramus osteotomy.

Background: Sagittal split ramus osteotomy is an established procedure used in oral and maxillofacial surgery. Major bleeding is representative intraoperative complication involves vessels such as the inferior alveolar, facial, and maxillary arteries and accompanying veins, in particular the retromandibular vein (RMV). We previously described the RMV course patterns using embalmed cadavers. However, owing to the possible influence of formalin fixation and unclear skeletal classification of the jaws, the present study aimed to use contrast-enhanced computed tomography to confirm the RMV in patients with defined jaw deformities. Methods: Twenty patients with orthognathic jaw deformities (40 sides) were included. The RMV and the lateral and posterior views of the mandible were evaluated using contrast-enhanced computed tomography. Course patterns and RMV positions were classified as previously reported. Results: Three patterns were identified in the lateral view. Type A: RMV posterior to the posterior border of the ramus (n=25; 62.5%). Type B: adjoining the posterior border of the ramus (n=12; 30.0%). Type C: anterior to the posterior border of the ramus (n=3; 7.5%). Five course patterns were identified in the posterior view. Pattern I: rectilinear course running medial to the posterior border of the ramus (n=3; 7.5%). Pattern II: diagonal course running medially from immediately posterior to posterior border of the ramus (n=11; 27.5%). Pattern III: rectilinear course running immediately posterior to the posterior border of the ramus (n=12; 30.0%). Pattern IV: diagonal course running from lateral to medial relative to the posterior border of the ramus (n=8; 20.0%). Pattern V: diagonal course running from lateral to immediately posterior relative to the posterior border of the ramus (n=6; 15.0%). In the no-course pattern group, the RMV inferior to the lingula was lateral to its position and superior to that of the lingula. In half of the cases, the left and right sides exhibited different running patterns. We observed no correlation between the skeletal patterns of jaw deformities and the course of the RMV. Conclusions: Type B/Pattern II that runs in a straight line bordering the posterior margin of the ramus requires the most attention during surgery. These findings suggest the possibility of predicting intraoperative bleeding risk.

Enhanced Precision in Genioplasty: A Novel Intraoperative Spatial Repositioning Using Computer-Aided Design and Manufacturing Technology and a Holographic Mixed Reality Application.

Genioplasty is performed for the orthognathic surgical correction of dentofacial deformities. This article reports a safe and accurate method for genioplasty combining a novel three-dimensional (3D) device with mixed reality (MR)-assisted surgery using a registration marker and a head-mounted display. Four types of devices were designed based on the virtual operation: a surgical splint with a connector; an osteotomy device; a repositioning device; and a registration marker. Microsoft HoloLens 2 and Holoeyes MD were used to project holograms created using computed tomography (CT) data onto the surgical field to improve the accuracy of the computer-aided designed and manufactured (CAD/CAM) surgical guides. After making an incision on the oral vestibule, the splint was fitted on the teeth and the osteotomy device was mounted at the junction site, placed directly on the exposed mandible bone surface. Temporary screws were fixed into the screw hole. An ultrasonic cutting instrument was used for the osteotomy. After separating the bone, a repositioning device was connected to the splint junction and bone segment, and repositioning was performed. At the time of repositioning, the registration marker was connected to the splint junction, and mandible repositioning was confirmed three-dimensionally through HoloLens 2 into the position specified in the virtual surgery. The rate of overlay error between the preoperative virtual operation and one-month postoperative CT data within 2 mm was 100%. CAD/CAM combined with MR enabled accurate genioplasty.

Structural analysis of the mylohyoid muscle as a septum dividing the floor of the oral cavity for the purposes of dental implant surgery: variety of muscle attachment positions and ranges of distribution.

OBJECTIVES: The objective was to investigate the details of the attachments of the mylohyoid muscle to the mandible anterior to the hyoid and mylohyoid lines to understand the positional relationship between the sublingual space and the mylohyoid, knowledge that is essential for dental implant surgery in the incisal region, as well as the routes of communication between the sublingual space and other spaces. METHODS: While evaluating the presence or absence of an anterior mylohyoid muscle fiber attachment to the mandible, sublingual gland herniation, spaces between muscle fascicles were also recorded as sites of penetration. The mean muscle thickness in each of these areas was also calculated. RESULTS: In all specimens, the mylohyoid originated not only from the mylohyoid line but also from the lingual surface of the center of the mandibular body (the mandibular symphysis) below the mental spines. The mylohyoid muscle fascicles were thickest in the posterior region, and further anterior to this, they tended to become thinner. Sublingual gland herniations passing through the mylohyoid were noted in the anterior and central regions, but not in the posterior region. Penetration between the muscle fascicles was most common in the central region, and no such penetration was evident in the posterior region. CONCLUSIONS: These results suggest that the mylohyoid functions only incompletely as a septum, and that routes of communication from the sublingual space to the submandibular space may be present in both the anterior and central muscle fascicles of the mylohyoid. Therefore, bleeding complications during dental implant placement in the anterior mandible can be serious issues. There is a potential for sublingual hematoma that could compromise the airway by pressing the tongue against the soft palate into the pharynx.

Increased RUNX3 expression mediates tumor-promoting ability of human breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major stromal component of human breast cancers and often promote tumor proliferation, progression and malignancy. We previously established an experimental CAF (exp-CAF) cell line equipped with a potent tumor-promoting ability. It was generated through prolonged incubation of immortalized human mammary fibroblasts with human breast cancer cells in a tumor xenograft mouse model. RESULTS: Herein, we found that the exp-CAFs highly express Runt-related transcription factor 3 (RUNX3), while counterpart fibroblasts do not. In breast cancer patients, the proportion of RUNX3-positive stromal fibroblast-like cells tends to be higher in cancerous regions than in non-cancerous regions. These findings suggest an association of RUNX3 with CAF characteristics in human breast cancers. To investigate the functional role of RUNX3 in CAFs, the exp-CAFs with or without shRNA-directed knockdown of RUNX3 were implanted with breast cancer cells subcutaneously in immunodeficient mice. Comparison of the resulting xenograft tumors revealed that tumor growth was significantly attenuated when RUNX3 expression was suppressed in the fibroblasts. Consistently, Ki-67 and CD31 immunohistochemical staining of the tumor sections indicated reduction of cancer cell proliferation and microvessel formation in the tumors formed with the RUNX3-suppressed exp-CAFs. CONCLUSION: These results suggest that increased RUNX3 expression could contribute to the tumor-promoting ability of CAFs through mediating cancer cell growth and neoangiogenesis in human breast tumors.

Mixed reality for extraction of maxillary mesiodens.

BACKGROUND: Mesiodentes are the most common supernumerary teeth. The cause is not fully understood, although proliferations of genetic factors and the dental lamina have been implicated. Mesiodentes can cause delayed or ectopic eruption of permanent incisors, which can further alter occlusion and appearance. Careful attention should be paid to the position and direction of the mesiodentes because of possible damage to adjacent roots in the permanent dentition period, errant extraction in the deciduous and mixed dentition periods, and damage to the permanent tooth embryo. To avoid these complications, we applied mixed reality (MR) technology using the HoloLens® (Microsoft, California). In this study, we report on three cases of mesiodentes extraction under general anesthesia using MR technology. RESULTS: The patients ranged in age from 6 to 11 years, all three were boys, and the direction of eruption was inverted in all cases. The extraction approach was palatal in two cases and labial in one case. The average operative time was 32 min, and bleeding was minimal in all cases. No intraoperative or postoperative complications occurred. An image was shared preoperatively with all the surgeons using an actual situation model. Three surgeons used Microsoft HoloLens® during surgery, shared MR, and operated while superimposing the application image in the surgical field. CONCLUSIONS: The procedure was performed safely; further development of MR surgery support systems in the future is suggested.

Clinical investigation of patients with jaw deformity with comorbidities.

BACKGROUND: With improvements in the safety and stability of surgeries, the number of orthognathic surgeries is increasing. Most patients who undergo orthognathic surgeries are younger, and the number of orthognathic surgeries for patients with comorbidities is also increasing. We report a survey and clinical investigation of patients with comorbidities who underwent orthognathic surgeries at our department to improve the safety of orthognathic surgery. RESULTS: The participants included 296 men and 712 women, with a mean age of 28 years (13-19 years, n=144; 20-29 years, n=483; 30-39 years, n=236; 40-49 years, n=102; 50-59 years, n=39; ≥60 years, n=4). In total, 347 patients underwent one-stage Le Fort type I osteotomy and sagittal split ramus osteotomy (SSRO), 243 underwent SSRO, 287 underwent plate removal, 126 underwent genioplasty and plate removal, and five underwent other surgeries. In total, 529 patients had comorbidities (52%), including allergic diseases (n=220, 33%), respiratory diseases (n=107, 16%), neurologic and psychiatric diseases (n=69, 10%), gynecologic diseases (n=28, 4%), hematologic diseases (n=27, 4%), cardiovascular diseases (n=24, 4%), digestive diseases (n=22, 3%), metabolic and endocrine diseases (n=18, 3%), spinal diseases (n=11, 2%), ophthalmologic diseases (n=11, 2%), renal and urological diseases (n=9, 1%), and other diseases (n=117, 18%). Among the patients with comorbidities, 11 with hemorrhagic diatheses (hemophilia and von Willebrand disease), arrhythmia (atrioventricular block), psychiatric disease (adjustment disorder), and metabolic disease (diabetes) required cautious perioperative management. The patient with hemophilia was managed with regular low-dose recombinant factor VIII replacement therapy, and the patient with type I diabetes mellitus was administered continuous insulin infusion and sliding-scale insulin therapy; both patients had an uneventful course. CONCLUSIONS: The study findings suggest that with the increase in orthognathic surgeries, oral and maxillofacial surgeons should adequately manage cases requiring cautious perioperative control and highlight the importance of preoperative screening. Despite the well-established safety and postoperative stability of orthognathic surgeries, oral surgeons should adopt appropriate additional preventive measures for patients with comorbidities.

A clinico-statistical study of factors associated with intraoperative bleeding in orthognathic surgery.

BACKGROUND: Excessive bleeding is a major intraoperative risk associated with orthognathic surgery. This study aimed to investigate the factors involved in massive bleeding during orthognathic surgeries so that safe surgeries can be performed. Patients (n=213) diagnosed with jaw deformities and treated with bimaxillary orthognathic surgery (Le Fort I osteotomy and bilateral sagittal split ramus osteotomy) in the Department of Oral and Maxillofacial Surgery at the Suidobashi Hospital, Tokyo Dental College between January 2014 and December 2016 were included. Using the patients' medical and operative records, the number of cases according to sex, age at the time of surgery, body mass index (BMI), circulating blood volume, diagnosis of maxillary deformity, direction of maxillary movement, operative duration, incidence of bad split, injury of nasal mucosa, and blood type were analyzed. RESULTS: The results revealed that BMI, circulating blood volume, nasal mucosal injury, and operative time were associated with the risk of intraoperative massive bleeding in orthognathic surgeries. Chi-square tests and binomial logistic regression analyses showed significant differences in BMI, circulating blood volume, direction of maxillary movement, operative duration, and injury to the nasal mucosa. Operative duration emerged as the most important risk factor. Furthermore, a >4-mm upward migration of the posterior nasal spine predicted the risk of massive bleeding in orthognathic surgery. CONCLUSIONS: The upward movement of the maxilla should be recognized during the preoperative planning stage as a risk factor for intraoperative bleeding, and avoiding damage to the nasal mucosa should be considered a requirement for surgeons to prevent massive bleeding during surgery.

Osteotomy training for dental students using three-dimensional simulation software and maxillofacial three-dimensional-printed models.

PURPOSE/OBJECTIVES: Simulated educational models of teeth, bones, and gums have been used for a long time in procedural skills training in dentistry.  The advent of three-dimensional (3D) printing technologies and additive manufacturing has facilitated the production of more advanced 3D printed models that can be utilized for surgical and dental training together with other technologies (e.g., 3D scanners and image analysis software). METHODS: We conducted training on osteotomy in the maxilla and mandible using maxillofacial simulation software (MSS) and a 3D-printed model for 5th-year undergraduate dental students (13 students in 2017 and 11 students in 2018 with more than half of their results). We compared the group (13 students) who performed osteotomy after participating in the simulation lecture with those (11 students) who performed osteotomy after performing self-simulation (they were instructed to bring their personal computers and install the MSS) using tests and questionnaires (pre- and post-curriculum). RESULTS: The average test score was significantly higher in the group who performed osteotomy using the 3D-printed model after performing self-simulation; participants in this group had acquired a better understanding of the surgical procedure. Comparison using questionnaires showed a significant difference in the students' understanding of instruments and surgical techniques between both groups. CONCLUSION: The MSS and 3D printed models are widely used clinically. Incorporating these in the curriculum will help accelerate student development. In addition, prompt education on the purpose and usefulness of these tools will not only facilitate simulation software and 3D-printed model-based treatment plan in the clinic but also promote further research.

Mixed reality and three dimensional printed models for resection of maxillary tumor: a case report.

In the field of oral and maxillofacial surgery, many institutions have recently begun using three-dimensional printers to create three-dimensional models and mixed reality in a variety of diseases. Here, we report the actual situation model which we made using three-dimensional printer from virtual operation data and the resection that was performed while grasping a maxillary benign tumor and neighboring three-dimensional structure by designing an application for Microsoft® HoloLens, and using Mixed Reality surgery support during the procedure.

A safe, stable, and convenient three-dimensional device for high Le Fort I osteotomy.

BACKGROUND: Le Fort I osteotomy is a highly effective treatment for skeletal jaw deformities and is commonly performed. High Le Fort I osteotomy is a modified surgical procedure performed for improving the depression of the cheeks by setting the osteotomy higher than the conventional Le Fort I osteotomy. Developments in three-dimensional (3D) technology have popularized the use of 3D printers in various institutions, especially in orthognathic surgeries. In this study, we report a safe and inexpensive method of performing a high Le Fort I osteotomy using a novel 3D device and piezosurgery, which prevent tooth root injury without disturbing the operation field for patients with a short midface and long tooth roots. RESULTS: A 17-year-old woman presented with facial asymmetry, mandibular protrusion, a short midface, and long tooth roots. We planned high Le Fort I osteotomy and bilateral sagittal split ramus osteotomy. Prevention of damage to the roots of the teeth and the infraorbital nerve and accurate determination of the posterior osteotomy line were crucial for clinical success. Le Fort I osteotomy using 3D devices has been reported previously but were particularly large in size for this case. Additionally, setting the fixing screw of the device was difficult, because of the risk of damage to the roots of the teeth. Therefore, a different surgical technique, other than the conventional Le Fort I osteotomy and 3D device, was required. The left and right parts of the 3D device were fabricated separately, to prevent any interference in the surgical field. Further, the 3D device was designed to accurately cover the bone surface from the piriform aperture to the infra-zygomatic crest with two fixation points (the anterior nasal spine and the piriform aperture), which ensured stabilization of the 3D device. The device is thin and does not interfere with the surgical field. Safe and accurate surgical performance is possible using this device and piezosurgery. The roots of the teeth and the infraorbital nerve were unharmed during the surgery. CONCLUSIONS: This device is considerably smaller than conventional devices and is a simple, low-cost, and efficient method for performing accurate high Le Fort I osteotomy.

Relationship between the immunohistological examination and fluorescence visualization of oral squamous cell carcinoma.

Disorders of the oral mucosa are considered easy to diagnose since they can be visualized and examined directly. A change in the color of the oral mucosa reflects histopathological changes and is an important diagnostic parameter. However, the subjective perception of color varies. To determine the extent of resection for oral mucosa conditions, it is necessary to digitize the color and perform objective assessments. In recent years, fluorescence visualization devices and analysis software that measure tissue luminance G have been employed for the identification of oral mucosa diseases. Fluorescence visualization is presumably based on the decrease in epithelial flavin adenine dinucleotide content and luminance G values due to the destruction of collagen cross-links [fluorescence visualization loss (FVL)]. However, cases with differences between luminance values and histopathological presentation exist. Therefore, additional factors may affect fluorescence visualization. The present study used a portable, non-contact oral mucosa fluorescence visualization device for luminance measurements in seven patients with oral squamous cell carcinoma. Furthermore, Picro-Sirius Red and immunohistochemical staining were performed for CK13, CK17, Ki67, p53 and E-cadherin in the FVL(+) (lesion) and FVL(-) (resection stump) areas to elucidate the principle of fluorescence visualization. Fluorescence was significantly lower in the FVL(+) than in the FVL(-) areas, and the mean luminance G value was 56. The Picro-Sirius Red stain revealed collagen destruction in the FVL(+) areas but no collagen disruption in the FVL(-) areas. CK13 was negative in the FVL(+) and positive in the FVL(-) areas, whereas the opposite pattern was observed for CK17. In the FVL(+) area, p53 staining was positive. E-cadherin expression was enhanced in the FVL(-) areas and reduced in the FVL(+) areas. Furthermore, the luminance G value tended to be lower in cases with weaker E-cadherin staining. The aforementioned results suggest that decreased E-cadherin expression may be a factor that regulates fluorescence visualization.

Maxillary reconstruction using tunneling flap technique with 3D custom-made titanium mesh plate and particulate cancellous bone and marrow graft: a case report.

BACKGROUND: Reconstructive surgery is often required for tumors of the oral and maxillofacial region, irrespective of whether they are benign or malignant, the area involved, and the tumor size. Recently, three-dimensional (3D) models are increasingly used in reconstructive surgery. However, these models have rarely been adapted for the fabrication of custom-made reconstruction materials. In this report, we present a case of maxillary reconstruction using a laboratory-engineered, custom-made mesh plate from a 3D model. CASE PRESENTATION: The patient was a 56-year-old female, who had undergone maxillary resection in 2011 for intraoral squamous cell carcinoma that presented as a swelling of the anterior maxillary gingiva. Five years later, there was no recurrence of the malignant tumor and a maxillary reconstruction was planned. Computed tomography (CT) revealed a large bony defect in the dental-alveolar area of the anterior maxilla. Using the CT data, a 3D model of the maxilla was prepared, and the site of reconstruction determined. A custom-made mesh plate was fabricated using the 3D model (Okada Medical Supply, Tokyo, Japan). We performed the reconstruction using the custom-made titanium mesh plate and the particulate cancellous bone and marrow graft from her iliac bone. We employed the tunneling flap technique without alveolar crest incision, to prevent surgical wound dehiscence, mesh exposure, and alveolar bone loss. Ten months later, three dental implants were inserted in the graft. Before the final crown setting, we performed a gingivoplasty with palate mucosal graft. The patient has expressed total satisfaction with both the functional and esthetic outcomes of the procedure. CONCLUSION: We have successfully performed a maxillary and dental reconstruction using a custom-made, pre-bent titanium mesh plate.

Novel condylar repositioning method for 3D-printed models.

BACKGROUND: Along with the advances in technology of three-dimensional (3D) printer, it became a possible to make more precise patient-specific 3D model in the various fields including oral and maxillofacial surgery. When creating 3D models of the mandible and maxilla, it is easier to make a single unit with a fused temporomandibular joint, though this results in poor operability of the model. However, while models created with a separate mandible and maxilla have operability, it can be difficult to fully restore the position of the condylar after simulation. The purpose of this study is to introduce and asses the novel condylar repositioning method in 3D model preoperational simulation. METHODS: Our novel condylar repositioning method is simple to apply two irregularities in 3D models. Three oral surgeons measured and evaluated one linear distance and two angles in 3D models. RESULTS: This study included two patients who underwent sagittal split ramus osteotomy (SSRO) and two benign tumor patients who underwent segmental mandibulectomy and immediate reconstruction. For each SSRO case, the mandibular condyles were designed to be convex and the glenoid cavities were designed to be concave. For the benign tumor cases, the margins on the resection side, including the joint portions, were designed to be convex, and the resection margin was designed to be concave. The distance from the mandibular ramus to the tip of the maxillary canine, the angle created by joining the inferior edge of the orbit to the tip of the maxillary canine and the ramus, the angle created by the lines from the base of the mentum to the endpoint of the condyle, and the angle between the most lateral point of the condyle and the most medial point of the condyle were measured before and after simulations. Near-complete matches were observed for all items measured before and after model simulations of surgery in all jaw deformity and reconstruction cases. CONCLUSIONS: We demonstrated that 3D models manufactured using our method can be applied to simulations and fully restore the position of the condyle without the need for special devices.

Metabolomic analysis of the saliva of Japanese patients with oral squamous cell carcinoma.

The aim of the present study was to characterize the metabolic systems in Japanese patients with oral squamous cell carcinoma (OSCC) using capillary electrophoresis-mass spectrometry (CE-MS) metabolome analysis of saliva samples. A previous study showed variations among ethnicities and tumor sites in the saliva metabolome of patients with OSCC using CE-MS. In the present study, saliva was obtained from 22 Japanese patients with OSCC and from 21 healthy controls who visited the Department of Dentistry, Oral and Maxillofacial Surgery, Tokyo Dental Collage Ichikawa General Hospital, Tokyo, Japan, and all samples were subject to comprehensive quantitative metabolome analysis using CE-MS. A total of 499 metabolites were detected as CE-MS peaks in the saliva tested from the two groups. A total of 25 metabolites were revealed as potential markers to discriminate between patients with OSCC and healthy controls: Choline, p-hydroxyphenylacetic acid, and 2-hydroxy-4-methylvaleric acid (P<0.001); valine, 3-phenyllactic acid, leucine, hexanoic acid, octanoic acid, terephthalic acid, γ-butyrobetaine, and 3-(4-hydroxyphenyl)propionic acid (P<0.01); and isoleucine, tryptophan, 3-phenylpropionic acid, 2-hydroxyvaleric acid, butyric acid, cadaverine, 2-oxoisovaleric acid, N6,N6,N6-trimethyllysine, taurine, glycolic acid, 3-hydroxybutyric acid, heptanoic acid, alanine, and urea (P<0.05, according to the Wilcoxon rank sum test). A previous study by Sugimoto and co-workers detected 24 discriminatory metabolites, 7 of which (taurine, valine, leucine, isoleucine, choline, cadaverine, and tryptophan) were also detected in the present study. In the present study, however, choline, metabolites in the branched chain amino acids (BCAA) cycle, urea, and 3-hydroxybutyric acid were also characterized. Choline and metabolites of the BCAA cycle have previously been reported in OSCC using metabolome analysis. To the best of our knowledge, no previous reports have identified urea and 3-hydroxybuyric acid in the metabolome of patients with OSCC. These findings suggest the usefulness of metabolites as salivary biomarkers for Japanese patients with OSCC. Further studies using larger patient cohorts should be conducted to validate these results.

Deletion polymorphism at chromosome 3q26.1 and oral squamous cell carcinoma.

Several recent studies have investigated DNA instability in malignancies including deletions and duplications of part of the chromosome using array-based comparative genomic hybridization (CGH) analysis. Using the same approach on oral squamous cell carcinoma (OSCC) tissue samples, we found a frequent deletion at chromosome 3q26.1 in OSCC patients; this polymorphism showed a gene frequency of 0.293-0.368 in healthy volunteers (n=60) and 0.129-0.195 in OSCC patients (n=54). Detailed analysis around the polymorphic region revealed the deletion breakage point. A significant association of gene frequency for the deletion polymorphism between healthy volunteers and patients implicated genetic factors related to this polymorphism in the development of OSCC. Currently, no gene is predicted to lie within the 3,606-kbp region around the polymorphism. Thus, although a single-gene model could not explain the occurrence of OSCC, we believe that examining this polymorphism could be useful in identifying risk factors for OSCC.

Copy number changes of CRISP3 in oral squamous cell carcinoma.

The aim of this study was to identify tumor suppressor genes (TSGs) in oral squamous cell carcinoma (OSCC) using whole-genome analysis of microarray technology and real-time quantitative polymerase chain reaction (QPCR). We applied whole-genome analysis of TSGs in the specimens from 3 patients of OSCC by microarray technology. A total of 11 genes, CRISP3, SCGB3A1, AGR2, PIP, C20orf114, TFF1, STATH, AZGP1, MUC7, DMBT1 and LOC389429, were found to be down-regulated, and 2, matrix metallopeptidase (MMP) 1 and MMP3, were found to be up-regulated in the 3 OSCC patients using microarray technology. In this study, we selected the CRISP3 gene. CRISP3 belongs to the cystein-rich secretary protein gene family in chromosome 6p12.3. CRISP3 has been found in the salivary gland, spleen and prostate gland and is a prominent biomarker in the gene expression of prostate cancer. Down-regulation of this gene was previously observed in OSCC. No studies examining the DNA copy number of CRISP3 in detail exist. We analyzed the DNA copy number of CRISP3 in 5 OSCC-derived cell lines (SAS, Ca9-22, KON, HSC2 and HSC4) and 60 OSCC tissues by real-time QPCR. The DNA copy number loss of CRISP3 was observed in 2 of the 5 OSCC-derived cell lines (SAS, HSC2) and in 24 of 60 patients (40.0%) using real-time QPCR. A significant statistical correlation between the copy number loss and gender and T classification was observed. These results indicate that the inactivation of CRISP3 is an early event in OSCC, since the T1/T2 classification is correlated with DNA copy number loss of CRISP3, whereas T3/T4 classification is not. We conclude that CRISP3 may be involved in the carcinogenesis of OSCC.