Loss of intercellular bridges in the depth of invasion measurement area is a novel negative prognostic factor for oral squamous cell carcinoma: A retrospective study.

OBJECTIVE: This study aimed to evaluate intercellular bridges in the depth of invasion (DOI) measurement area as prognostic factors in oral squamous cell carcinoma (OSCC). STUDY DESIGN: The mode of invasion was determined based on the Yamamoto-Kohama classification system by observing the hematoxylin-eosin-stained whole-slide images of specimens obtained from 78 patients with OSCC, and the clinicopathologic features were characterized. The presence of intercellular bridges was analyzed in 46 patients with Yamamoto-Kohama classification grade ≥3 whose DOI was measured by dividing the measurement area into 3 parts: the surface, center, and front of the tumor. RESULTS: Univariate analyses identified lymph node metastasis, loss of intercellular bridges in the DOI measurement area, DOI of ≥4500 µm, and pattern of invasion 4C-4D as negative prognostic factors. Multivariate analyses revealed that lymph node metastasis and the loss of intercellular bridges in the entire area were independent factors, with hazard ratios of 9.34 (95% confidence interval, 2.09-42.03; P = .003) and 3.64 (95% confidence interval, 1.10-11.99; P = .045), respectively. CONCLUSIONS: Loss of intercellular bridges in the DOI measurement area is a negative prognostic factor for OSCC and may be useful in selecting treatment.

Ets family proteins regulate the EMT transcription factors Snail and ZEB in cancer cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during epithelial tumor progression. Snail and ZEB1/2 (ZEB1 and ZEB2), known as EMT transcription factors, are key regulators of this transition. ZEB1/2 are positively correlated with EMT phenotypes and the aggressiveness of cancers. On the contrary, Snail is also correlated with the aggressiveness of cancers, but is not correlated with the expression of EMT marker proteins. Snail is induced by transforming growth factor-ß (TGF-ß), a well-known inducer of EMT, in various cancer cells. Interestingly, Snail induction by TGF-ß is markedly enhanced by active Ras signals. Thus, cancer cells harboring an active Ras mutation exhibit a drastic induction of Snail by TGF-ß alone. Here, we found that members of the E26 transformation-specific (Ets) transcription factor family, Ets1 and Ets2, contribute to the upregulation of both Snail and ZEB1/2. Snail induction by TGF-ß and active Ras is dramatically inhibited using siRNAs against both Ets1 and Ets2 together, but not on their own; in addition, siRNAs against both Ets1 and Ets2 also downregulate the constitutive expression of Snail and ZEB1/2 in cancer cells. Examination of several alternatively spliced variants of Ets1 revealed that p54-Ets1, which includes exon VII, but not p42-Ets1, which excludes exon VII, regulates the expression of the EMT transcription factors, suggesting that Ets1 is a crucial molecule for regulating Snail and ZEB1/2, and thus cancer progression is mediated through post-translational modification of the exon VII domain.

High expression of protein tyrosine kinase 7 in oral squamous cell carcinoma: Clinicopathological correlation and prognosis relevance.

BACKGROUND: The purpose of this study was to evaluate the association between the　immunohistochemistry (IHC) of protein tyrosine kinase 7 (PTK7) expression and clinicopathological factors of oral squamous cell carcinoma (OSCC). METHODS: Tissue specimens were obtained from 80 patients with primary OSCC. IHC scoring was conducted according to the rate of positive cell and staining intensity. We used the IHC score to classify the degree of PTK7 expression and evaluate clinicopathological factors and prognosis. RESULTS: The number of the high expression group (IHC Score 2 or 3) was 45 cases and that of the low expression group (IHC Score 0 or 1) was 35 cases. A significant difference between high expression and low expression groups was found in the N category (p = .008), degree of differentiation (p < .001), and pattern of invasion (p < .001). In accordance with the exacerbation of OSCC with respect to three parameters (N category, degree of differentiation, and pattern of invasion), the ratio of high expression of PTK7 increased. The overall 5-year survival rate was 59.3% in the high expression group and 87.3% in the low expression group (p < .05). The pathological prognostic signs affecting overall survival were evaluated by univariate analysis and multivariate analysis with Cox proportional hazards model and showed an association with lymph node metastasis and invasion patterns. CONCLUSION: This study suggests that a high IHC score of PTK7 is a potential biomarker for predicting potential metastasis.

Automatic discrimination of Yamamoto-Kohama classification by machine learning approach for invasive pattern of oral squamous cell carcinoma using digital microscopic images: a retrospective study.

OBJECTIVE: The Yamamoto-Kohama criteria are clinically useful for determining the mode of tumor invasion, especially in Japan. However, this evaluation method is based on subjective visual findings and has led to significant differences in determinations between evaluators and facilities. In this retrospective study, we aimed to develop an automatic method of determining the mode of invasion based on the processing of digital medical images. STUDY DESIGN: Using 101 digitized photographic images of anonymized stained specimen slides, we created a classifier that allowed clinicians to introduce feature values and subjected the cases to machine learning using a random forest approach. We then compared the Yamamoto-Kohama grades (1, 2, 3, 4C, 4D) determined by a human oral and maxillofacial surgeon with those determined using the machine learning approach. RESULTS: The input of multiple test images into the newly created classifier yielded an overall F-measure value of 87% (grade 1, 93%; grade 2, 67%; grade 3, 89%; grade 4C, 83%; grade 4D, 94%). These results suggest that the output of the classifier was very similar to the judgments of the clinician. CONCLUSIONS: This system may be valuable for diagnostic support to provide an accurate determination of the mode of invasion.

Assessment of lateral pterygoid muscle and temporomandibular joint disc after Le Fort I osteotomy with and without intentional pterygoid plate fracture and sagittal split ramus osteotomy in class II and class III patients.

The aim of the study was to examine lateral pterygoid muscle (LPM) and temporomandibular joint (TMJ) disc before and after Le Fort I osteotomy with and without intentional pterygoid plate fracture and sagittal split ramus osteotomy (SSRO) in class II and class III patients. Le Fort I osteotomy and SSRO were performed in class II and class III patients. LPM measurements using oblique sagittal computed tomography (CT) images and TMJ disc position using magnetic resonance imaging (MRI) were examined. Statistical comparisons were performed for the LPM and TMJ between class II and class III patients and between those with and without intentional pterygoid plate fracture in Le Fort I osteotomy. The subjects comprised 60 female patients (120 sides), with 30 diagnosed as class II and 30 as class III. Preoperatively, the width of the condylar attachment, width at eminence, length of the LPM, angle of the LPM, and square of the LPM were significantly smaller in the class II group than in the class III group (p < 0.05). After 1 year, the width of the condylar attachment, width at eminence, and angle of the LPM remained significantly smaller in the class II group than in the class III group (p < 0.0001). TMJ disc position was significantly related to the width of the condylar attachment of the LPM, both pre- and postoperatively (p < 0.0001). However, postoperative disc position did not change in all patients. Next, the class II patients (60 sides) were divided into two groups who underwent Le Fort I osteotomy with or without intentional pterygoid plate fracture. Changes in all measurements of the LPM showed no significant differences between these two groups. Our study suggested that TMJ disc position classification could be associated with the width of condylar attachment of the LPM before and after surgery, while the surgical procedure, including Le Fort I osteotomy with intentional pterygoid plate fracture, might not affect postoperative LMP or disc position in class II patients.

Computed tomography assessment of mandibular morphologic changes and the inferior mandibular border defect after sagittal split ramus osteotomy.

OBJECTIVE: This study aimed to assess mandibular morphologic changes to the condyle, ramus, mandibular body, and inferior mandibular border defect after sagittal split ramus osteotomy in class II and III patients. STUDY DESIGN: The relationships among the condyle, ramus, and mandibular body measured by computed tomography preoperatively and postoperatively were assessed and factors related to the reduction of the condylar square and mandibular inferior border defect were examined. RESULTS: Patients included 72 female patients with jaw deformity (36 skeletal class II cases, 36 skeletal class III cases). Postoperative reduction of the condylar square was significantly correlated with preoperative condylar height in patients with class II (P = .0297) vs preoperative condylar height and preoperative mandibular height in patients with class III (P < .0001). A mandibular inferior border defect was found in 18 of 72 class II sides (25.0%) and was significantly related to the position of the osteotomy line and attachment side of the inferior border cortex (P < .0001). CONCLUSIONS: This study's findings suggest that the postoperative reduction of the condyle could be associated with preoperative condylar height. However, the mandibular inferior border defect in class II advancement surgery could be independently associated with technical factors in sagittal split ramus osteotomy.

Evaluation of condylar surface CT values related to condylar height reduction after orthognathic surgery.

This study was performed to evaluate the relationship between condylar height reduction and changes in condylar surface computed tomography (CT) values in jaw deformity patients following orthognathic surgery. Mandibular advancement by sagittal split ramus osteotomy (SSRO) with Le Fort I osteotomy was performed in class II patients, and mandibular setback by SSRO with Le Fort I osteotomy was performed in class III patients. The maximum CT values (pixel values) at five points on the condylar surface and the condylar height, ramus height, condylar square, ramus angle, and gonial angle in the sagittal plane were measured preoperatively and 1 year postoperatively. Disc position was classified as anterior disc displacement (ADD) or other types by using magnetic resonance imaging (MRI). Ninety-two condyles of 46 female patients were prepared for this study. Their temporomandibular joints (TMJs) were divided into two groups based on class (46 joints in class II and 46 joints in class III) and two groups based on the findings (25 joints with ADD and 67 joints with other findings). ADD with and without reduction was observed in two joints in the class III group and in 23 joints in the class II group. The distribution of ADD incidence had not changed 1 year after surgery. Condylar height decreased 1 year after surgery in both class II patients (mandibular advancement) (p < 0.0001) and class III patients (mandibular setback) (p = 0.0306). Similarly, condylar height decreased 1 year after surgery both in patients who showed ADD (p = 0.0087) and those with other types (p = 0.0023). Significant postoperative increases at all angle sites on the condylar surface were found in the class II (p < 0.05) and ADD (p < 0.05) groups. This study showed that an enhanced condylar surface CT value might be one sign of condylar height reduction related to sequential condylar resorption, in combination with ADD.

EHF suppresses cancer progression by inhibiting ETS1-mediated ZEB expression.

ETS homologous factor (EHF) belongs to the epithelium-specific subfamily of the E26 transformation-specific (ETS) transcription factor family. Currently, little is known about EHF's function in cancer. We previously reported that ETS1 induces expression of the ZEB family proteins ZEB1/δEF1 and ZEB2/SIP1, which are key regulators of the epithelial-mesenchymal transition (EMT), by activating the ZEB1 promoters. We have found that EHF gene produces two transcript variants, namely a long form variant that includes exon 1 (EHF-LF) and a short form variant that excludes exon 1 (EHF-SF). Only EHF-SF abrogates ETS1-mediated activation of the ZEB1 promoter by promoting degradation of ETS1 proteins, thereby inhibiting the EMT phenotypes of cancer cells. Most importantly, we identified a novel point mutation within the conserved ETS domain of EHF, and found that EHF mutations abolish its original function while causing the EHF protein to act as a potential dominant negative, thereby enhancing metastasis in vivo. Therefore, we suggest that EHF acts as an anti-EMT factor by inhibiting the expression of ZEBs, and that EHF mutations exacerbate cancer progression.

Role of Platelet C-Type Lectin-Like Receptor 2 in Promoting Lung Metastasis in Osteosarcoma.

The overall prognosis of patients with sarcoma-based cancers has changed little in the last 20 years. There is an urgent need to investigate the metastatic potential of these tumors and to develop anti-metastatic drugs. It is becoming increasingly clear that platelets play an important role in the establishment of metastasis of carcinoma cells and could be a useful therapeutic target for patients with carcinoma. However, little is known about the role of platelets in sarcoma progression. Here, we investigated how osteosarcoma progression relates to platelet function to explore the possibility of anti-platelet therapy. We found that, similar to carcinoma cells, podoplanin (also known as Aggrus)-positive osteosarcoma cells induce platelet aggregation and activation. Administration of anti-glycoprotein Ibα (GPIbα, also known as CD42b) antibody reduced the lung metastasis of osteosarcoma. The supernatant from platelets cocultured with osteosarcoma cells contained several growth factors and promoted proliferation, invasiveness, and sphere formation of osteosarcoma cells in vitro. In addition, the development of lung metastasis was highly dependent on direct interaction between osteosarcoma cells and platelets. To explore the therapeutic target, we focused on the interactions between podoplanin on osteosarcoma and C-type lectin-like receptor (CLEC)-2 on platelets. The administration of a depleting antibody against CLEC-2 efficiently suppressed osteosarcoma metastasis into the lung. We also analyzed clinical data from patient samples at primary and metastatic sites. Although GPIbα expression was similar between the two sites, there was a significant increase in podoplanin at the metastatic site compared to that in the primary site, and the level of podoplanin expression in the primary site correlated with patient prognosis. These findings suggest that blockade of interactions between platelets CLEC-2 and osteosarcoma podoplanin represent the most promising therapeutic strategy for preventing the lung metastasis of osteosarcoma. © 2020 American Society for Bone and Mineral Research.

Addiction of mesenchymal phenotypes on the FGF/FGFR axis in oral squamous cell carcinoma cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during epithelial tumor progression. ZEB1/2 are EMT transcription factors that are positively correlated with EMT phenotypes and breast cancer aggressiveness. ZEB1/2 regulate the alternative splicing and hence isoform switching of fibroblast growth factor receptors (FGFRs) by repressing the epithelial splicing regulatory proteins, ESRP1 and ESRP2. Here, we show that the mesenchymal-like phenotypes of oral squamous cell carcinoma (OSCC) cells are dependent on autocrine FGF-FGFR signaling. Mesenchymal-like OSCC cells express low levels of ESRP1/2 and high levels of ZEB1/2, resulting in constitutive expression of the IIIc-isoform of FGFR, FGFR(IIIc). By contrast, epithelial-like OSCC cells showed opposite expression profiles for these proteins and constitutive expression of the IIIb-isoform of FGFR2, FGFR2(IIIb). Importantly, ERK1/2 was constitutively phosphorylated through FGFR1(IIIc), which was activated by factors secreted autonomously by mesenchymal-like OSCC cells and involved in sustained high-level expression of ZEB1. Antagonizing FGFR1 with either inhibitors or siRNAs considerably repressed ZEB1 expression and restored epithelial-like traits. Therefore, autocrine FGF-FGFR(IIIc) signaling appears to be responsible for sustaining ZEB1/2 at high levels and the EMT phenotype in OSCC cells.

Mosaicism of an ELANE mutation in an asymptomatic mother in a familial case of cyclic neutropenia.

PURPOSE: To confirm and characterize mosaicism of the cyclic neutropenia (CyN)-related mutation in the ELANE gene identified in the asymptomatic mother of patients with CyN. METHODS: We identified sibling cases with CyN due to a novel heterozygous splicing site mutation, IVS4 +5SD G>T, in the ELANE gene, resulting in an internal in-frame deletion of 30 nucleotides (corresponding to a ten amino acid deletion, V161-F170). The mutated allele was also detected in their asymptomatic mother but at low frequency. We measured the frequency of the mutant allele from peripheral blood leukocytes (PBLs) by subcloning, and confirmed the allelic frequency of mosaicism in various cell types by massively parallel DNA sequencing (MPS) analysis. RESULTS: In the subcloning analysis, the mutant allele was identified in 21.36 % of PBLs from the asymptomatic mother, compared with 54.72 % of PBLs from the CyN patient. In the MPS analysis, the mutant allele was observed in approximately 30 % of mononuclear cells, CD3(+) T cells, CD14(+) monocytes and the buccal mucosa. Conversely, it was detected in low frequency in polymorphonuclear leukocytes (PLMLs) (3-4 %) and CD16(+) granulocytes (2-3 %). CONCLUSIONS: Mosaicism of the ELANE mutation has only previously been identified in one confirmed and one unconfirmed case of SCN. This is the first report of mosaicism of the ELANE mutation in a case of CyN. The MPS results suggest that this de novo mutation occurred during the two-cell stage of embryogenesis. PLMLs expressing the ELANE mutation were found to be actively undergoing apoptosis.