Anti-VEGFR2 neutralising antibody slows the progression of multistep oral carcinogenesis.

Angiogenesis plays an important role in cancer growth and metastasis, and it is considered a therapeutic target to control tumour growth following anti-angiogenic therapy. However, it is still unclear when tissues initiate angiogenesis during malignant transformation from premalignant condition and whether this premalignant condition could be a therapeutic target of anti-angiogenic therapy. In this study, we aimed to analyse the onset of angiogenesis by evaluating morphological and functional alterations of microvessels during oral multistep carcinogenesis using a 4-nitroquinoline 1-oxide (4NQO)-induced oral carcinogenesis mouse model. In the study, we initially confirmed that with the use of 4NQO, oral lesions develop in a stepwise manner from normal mucosa through oral epithelial dysplasia (OED) to oral squamous cell carcinoma (OSCC). Evaluation of CD31-immunostained specimens revealed that microvessel density (MVD) increases in a stepwise manner from OEDs. Histological and functional analyses revealed the structural abnormalities and leakage of blood vessels had already taken place in OED. Then we evaluated the expression profiles of Hif1a and Vegfa along with hypoxic status and found that OED exhibited increased Vegfa expression under hypoxic conditions. Finally, we tested the possibility of OEDs as a target of anti-angiogenic therapy and found that anti-VEGFR2 neutralising antibody in OED slowed the disease progression from OED to OSCC. These data indicate that an angiogenic switch occurs at the premalignant stage and morphological, and functional alterations of microvessels already exist in OED. These findings also elucidate the tumour microenvironment, which gradually develops along with carcinogenic processes, and highlight usefulness of the 4NQO-induced carcinogenesis model in the study of epithelial and stromal components, which will support epithelial carcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Biological and clinical significance of the YKL-40/osteopontin-integrin ß4-p70S6K axis induced by macrophages in early oesophageal squamous cell carcinoma.

M2 macrophages contribute to the progression of oesophageal squamous cell carcinoma (ESCC); however, the roles of M2 macrophages in early ESCC remain unclear. To clarify the biological mechanisms underlying the interaction between M2 macrophages and oesophageal epithelial cells in early-stage ESCC, in vitro co-culture assays between the immortalised oesophageal epithelial cell line Het-1A and cytokine-defined M2 macrophages were established. Co-culture with M2 macrophages promoted the proliferation and migration of Het-1A cells via the mTOR-p70S6K signalling pathway activated by YKL-40, also known as chitinase 3-like 1, and osteopontin (OPN) that were hypersecreted in the co-culture supernatants. YKL-40 and OPN promoted the above phenotypes of Het-1A by making a complex with integrin ß4 (ß4). Furthermore, YKL-40 and OPN promoted M2 polarisation, proliferation, and migration of macrophages. To validate the pathological and clinical significances of in vitro experimental results, immunohistochemistry of human early ESCC tissues obtained by endoscopic submucosal dissection (ESD) was performed, confirming the activation of the YKL-40/OPN-ß4-p70S6K axis in the tumour area. Moreover, epithelial expression of ß4 and the number of epithelial and stromal infiltrating YKL-40- and OPN-positive cells correlated with the Lugol-voiding lesions (LVLs), a well-known predictor of the incidence of metachronous ESCC. Furthermore, the combination of high expression of ß4 and LVLs or high numbers of epithelial and stromal infiltrating YKL-40- and OPN-positive immune cells could more clearly detect the incidence of metachronous ESCC than each of the parameters alone. Our results demonstrated that the YKL-40/OPN-ß4-p70S6K axis played important roles in early-stage ESCC, and the high expression levels of ß4 and high numbers of infiltrating YKL-40- and OPN-positive immune cells could be useful predictive parameters for the incidence of metachronous ESCC after ESD. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Rab32 and Rab38 maintain bone homeostasis by regulating intracellular traffic in osteoclasts.

Osteoclasts play a crucial role in bone homeostasis by forming resorption pits on bone surfaces, resulting in bone resorption. The osteoclast expression of Rab38 protein is highly induced during differentiation from macrophages. Here we generated mice with double knockout (DKO) of Rab38 and its paralogue, Rab32, to investigate the roles of these proteins in osteoclasts. Bone marrow-derived macrophages from Rab32/38 DKO mice differentiated normally into osteoclasts in vitro. However, DKO osteoclasts showed reduced bone resorption activity. These osteoclasts also demonstrated defective secretion of tartrate-resistant acid phosphatase and cathepsin K into culture medium. Furthermore, the plasma membrane localization of a3, an osteoclast-specific a subunit of V-ATPase, was abrogated in DKO mice, substantiating the reduced resorption activity. In vivo, Rab32- and Rab38-positive cells were attached to the bone surface. Eight-week-old DKO mice showed significantly thickened trabecular bones in micro-CT and histomorphometry analysis, as well as reduced serum levels of cross-linked C-telopeptide of type I collagen, indicating diminished bone resorption in vivo. In DKO male mice over 10 weeks of age, hyperostosis appeared at the talofibular syndesmosis, the distal junction of the tibia and fibula. Furthermore, middle-aged mice (10 to 12 months of age) exhibited kyphosis, which is not usually observed in wild-type male mice until around 24 months of age. These results indicate that Rab32 and Rab38 contribute to osteoclast function by supporting intracellular traffic, thereby maintaining normal bone homeostasis.Key words: Rab32, Rab38, osteoclast, lysosome-related organelle, secretory lysosome.

The Germinal Origin of Salivary and Lacrimal Glands and the Contributions of Neural Crest Cell-Derived Epithelium to Tissue Regeneration.

The vertebrate body comprises four distinct cell populations: cells derived from (1) ectoderm, (2) mesoderm, (3) endoderm, and (4) neural crest cells, often referred to as the fourth germ layer. Neural crest cells arise when the neural plate edges fuse to form a neural tube, which eventually develops into the brain and spinal cord. To date, the embryonic origin of exocrine glands located in the head and neck remains under debate. In this study, transgenic TRiCK mice were used to investigate the germinal origin of the salivary and lacrimal glands. TRiCK mice express fluorescent proteins under the regulatory control of Sox1, T/Brachyury, and Sox17 gene expressions. These genes are representative marker genes for neuroectoderm (Sox1), mesoderm (T), and endoderm (Sox17). Using this approach, the cellular lineages of the salivary and lacrimal glands were examined. We demonstrate that the salivary and lacrimal glands contain cells derived from all three germ layers. Notably, a subset of Sox1-driven fluorescent cells differentiated into epithelial cells, implying their neural crest origin. Also, these Sox1-driven fluorescent cells expressed high levels of stem cell markers. These cells were particularly pronounced in duct ligation and wound damage models, suggesting the involvement of neural crest-derived epithelial cells in regenerative processes following tissue injury. This study provides compelling evidence clarifying the germinal origin of exocrine glands and the contribution of neural crest-derived cells within the glandular epithelium to the regenerative response following tissue damage.

Stage- and tissue-specific effect of cyclophosphamide during tooth development.

OBJECTIVE: The aim of this study was to investigate the toxic effect of cyclophosphamide (CPA) in the development of rodent molars. METHODS: CPA was administered intraperitoneally in postnatal mice between Day 1 and Day 10, and the morphological phenotype was evaluated at Day 26 using micro-computed tomography and histological analysis, including cell proliferation and cell death analyses. RESULTS: M3 molars of the mice who received 100 mg/kg CPA treatment at Day 6 or M2 molars who received treatment at Day 1 resulted in tooth agenesis or marked hypoplasia. Histological observation demonstrated that CPA treatment at Day 6 resulted in shrinkage of the M3 tooth germs, with a significant reduction in the proliferation of apoptotic cells. Conversely, CPA exposure at Day 2, which occurs at around the bud stage of M3, resulted in crown and root hypoplasia, with reduced numbers of cusp and root. In addition, CPA exposure at Day 10, which is the late bell stage of M3, induced root shortening; however, it did not affect crown morphogenesis. LIMITATIONS: The timing of CPA administration is limited to after birth. Therefore, its effect during the early stages of M1 and M2 could not be investigated. CONCLUSION: Defective phenotypes were evident in both crown and roots due to the effect of CPA. Interestingly, the severity of the phenotypes was associated with the developmental stages of the tooth germs at the time of CPA administration. The cap/early bell stage is the most susceptive timing for tooth agenesis, whereas the late bell stage is predominantly affected in terms of root formation by CPA administration.

Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.

Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.

Wnt signaling in cartilage development and diseases: lessons from animal studies.

Cartilage not only plays essential roles in skeletal development and growth during pre- and postnatal stages but also serves to provide smooth movement of skeletons throughout life. Thus, dysfunction of cartilage causes a variety of skeletal disorders. Results from animal studies reveal that ß-catenin-dependent canonical and independent non-canonical Wnt signaling pathways have multiple roles in regulation of cartilage development, growth, and maintenance. ß-Catenin-dependent signaling is required for progression of endochondral ossification and growth of axial and appendicular skeletons, while excessive activation of this signaling can cause severe inhibition of initial cartilage formation and growth plate organization and function in mice. In contrast, non-canonical Wnt signaling is important in columnar organization of growth plate chondrocytes. Manipulation of Wnt signaling causes or ameliorates articular cartilage degeneration in rodent osteoarthritis models. Human genetic studies indicate that Wnt/ß-catenin signaling is a risk factor for osteoarthritis. Accumulative findings from analysis of expression of Wnt signaling molecules and in vivo and in vitro functional experiments suggest that Wnt signaling is a therapeutic target for osteoarthritis. The target tissues of Wnt signaling may be not only articular cartilage but also synovium and subchondral bone.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation.

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis-Sox9, Sox6, and Sox5-were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen-cartilage-specific ECM substances-were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.

Melanotic oncocytic metaplasia of the nasopharynx.

We present three cases of melanotic oncocytic metaplasia of the nasopharynx. Case 1 and Case 2 were a 70- and a 61-year-old woman, and Case 3 was a 74-year-old man. Although Case 1 was asymptomatic, Cases 2 and 3 had hoarseness. All cases were Japanese and their nasopharyngeal examination revealed single or multiple black nodules measuring a few millimeters in diameter. In each case, biopsies were performed to rule out malignancy. Histological examination showed respiratory mucosa with oncocytic metaplasia and melanin pigments. Immunohistochemically, S-100 protein and melan-A positive dendritic melanocytes were observed in the basal layer of the oncocytes. Melanotic oncocytic metaplasia is extremely rare, and so far only 21 cases have been reported in the English literature to our knowledge. It has been reported in only older Asians, predominantly in males; there have been only three female patients including our two cases. All of our cases were long-time smokers, which supports the previously described hypothesis that smoking may be a predisposing factor for melanin pigmentation. Since melanotic oncocytic metaplasia may clinically mimic a malignant tumor, it is important to be aware of this lesion.

Prognostic impact of p16 and p53 expression in oropharyngeal squamous cell carcinomas.

BACKGROUNDS: A p16 protein is known to be overexpressed in human papillomavirus-positive head and neck squamous cell carcinoma specimens. p53 is a tumor suppressor protein detectable by immunohistochemistry in carcinogen-associated head and neck squamous cell carcinoma as a result of gene mutations. The purpose of this study is to investigate the prognostic impact of p16 and p53 expression in oropharyngeal squamous cell carcinomas. METHODS: We retrospectively examined the relationship between prognosis, and p16 and p53 expression levels of oropharyngeal squamous cell carcinoma specimens in 53 patients using immunohistochemistry. RESULTS: Overall, 55% of patients were p16 positive and 45% p16 negative, while 28% were p53 positive and 72% p53 negative. The p16 status showed an inverse relationship with the p53 status. A survival analysis by p16 status, p53 status, Union for International Cancer Control stage and main treatment modality demonstrated that only p16 status was related to better prognosis in terms of overall survival and disease-specific survival (3-year overall survival, 87 vs. 62%, P = 0.02; 3-year disease-specific survival, 90 vs. 62%, P = 0.02). To evaluate the practical prognostic factors in oropharyngeal squamous cell carcinoma patients, we classified patients as either p16-positive or p53-negative oropharyngeal squamous cell carcinomas, representing human papillomavirus-related oropharyngeal squamous cell carcinoma with wild-type p53 or the remaining patients with p16-negative or p53-positive OPSCCs, respectively. The former group showed survival advantages in terms of overall survival and disease-specific survival by log-tank test compared with the latter group (3-year overall survival, 96 vs. 58%, P = 0.005; 3-year disease-specific survival, 96 vs. 63%, P = 0.02). CONCLUSIONS: A group of patients who were p16 positive/p53 negative had better prognoses in terms of overall survival and disease-specific survival than that who were p16-positive alone.

A case of diffuse infiltrating gastrointestinal stromal tumor of sigmoid colon with perforation.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and typically present as discrete well-circumscribed but non-encapsulated tumor masses. In this report, we describe a case of colonic perforation caused by an unusual form of GIST. A 72-year-old Japanese woman presented to the emergency department with acute abdominal pain. Under the provisional diagnosis of sigmoid colon perforation, a laparoscopic sigmoidectomy was performed. Although the tumor mass was undetectable during the preoperative examination, a spindle cell lesion with a diffuse longitudinal growth pattern replacing the muscularis propria was revealed by microscopic examination. The spindle cell lesion was exposed at the perforation, suggesting a causal relationship between the lesion and the perforation. The spindle cell lesion was KIT-positive and had a mutation in the C-KIT gene at exon 11. We diagnosed it as diffuse infiltrating GIST. We consider that the lesion would be a cause of the colonic perforation, and emphasize the importance of accurate diagnosis of the lesion by histological, immunohistochemical and genetic examinations.

[A case of intraorbital solitary fibrous tumor resected successfully with preoperative arterial embolization].

The solitary fibrous tumor (SFT) is a rare spindle cell neoplasm derived from mesenchymal cells. It sometimes recurs clinically, and is categorized as an 'intermediate malignancy' tumor under the WHO (World Health Organization) classification of soft tissue tumors. Several studies have reported on intraorbital SFTs, but none of them has pointed out the utility of preoperative arterial embolization in the case of an intraorbital SFT. A 75-year-old man, who had received a dacryocystectomy for a benign tumor in the right lacrimal sac 30 years previously, visited our hospital complaining of lower eyelid swelling and lachrymation that had persisted for a year. CT and MRI revealed an intraorbital lesion, and the open biopsy specimen showed dense growth of spindle cells, which turned out to be an SFT by immunohistochemistry based on the findings. After preoperative embolization of the infraorbital artery, we removed the tumor with a skin incision on the lower rim of the orbit with little bleeding. The surgical specimen revealed that the tumor was close to a lacrimal canaliculus, which suggested the tumor originated from the lacrimal apparatus considering the patient's past history. He was followed up for three months without recurrences.

Macrocystic secretory carcinoma arising from the buccal minor salivary gland clinically mimicking a mucocele: A case report.

Secretory carcinoma (SC) is an uncommon salivary gland tumor that has been recently conceptualized. The present report describes a case of SC that was diagnosed as a mucocele on preoperative examination. A 46-year-old man presented to the Department of Oral and Maxillofacial Surgery at Saiseikai Senri Hospital (Suita-shi, Japan) with a main complaint of swelling of the right buccal mucosa. A mobile, elastic, hard mass was found beneath the right normal-appearing buccal mucosa. T2-weighted magnetic resonance imaging revealed a well-defined, internally homogeneous high-signal area with a maximum diameter of 18 mm. Based on the clinical diagnosis of mucocele, the buccal lesion was excised. Histopathological, immunohistochemical and fluorescence in situ hybridization analyses revealed the cystic lesion to be a macrocystic SC of a minor salivary gland. SC may have a mucocele-like appearance on magnetic resonance imaging. Even if a non-neoplastic lesion is suspected, the possibility of a malignant lesion such as SC must be considered for salivary gland disease.

Intercellular adhesion molecule-1 (ICAM-1) expression correlates with oral cancer progression and induces macrophage/cancer cell adhesion.

Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein in the immunoglobulin superfamily, which plays an important role in cell adhesion and signal transduction. Although ICAM-1 is believed to play a role in several malignancies, it is still uncertain whether or not ICAM-1 expression contributes to cancer progression. In this study, we performed clinicopathological and cell biological analyses of ICAM-1 expression in oral squamous cell carcinoma (SCC). First, we examined the ICAM-1 expression in tongue SCC immunohistochemically, and revealed that ICAM-1 was expressed predominantly at the invasive front area of tongue SCC. ICAM-1 expression at the invasive front area was correlated with invasion, lymph node metastasis and increased blood and lymphatic vessel density of the tongue SCC. The relationship between ICAM-1 expression and clinicopathological factors were consistent with the increased proliferation, invasion and cytokine-production activities of ICAM-1-transfected SCC cells. Second, we analyzed the relationship between macrophages and ICAM-1-expressing tongue SCC cells because ICAM-1 is known to act as a ligand for adhesion of immune cells. Increased ICAM-1 expression in tongue SCC was correlated with increased macrophage infiltration within SCC nests. Moreover, macrophage/SCC-cell adhesion through ICAM-1 molecule was revealed using an in vitro cell adhesion and blockade assay. These findings indicate that ICAM-1 plays an important role in tongue SCC progression, which may result from the SCC-cell activity, angiogenic activity, lymphangiogenic activity and macrophage/SCC-cell adhesion.

Mild obesity reduces survival and adiponectin sensitivity in endotoxemic rats.

BACKGROUND: Recent meta-analyses have reported that critically ill patients with morbid obesity (body mass index >40 kg/m(2)) have poor outcomes, but the effects and mechanisms of action of mild obesity are still unclear. The purpose of this study was to evaluate the effect of mild obesity using a lard-based, high-fat diet (HFD) on pathologic conditions and the mechanisms of adiponectin action in endotoxemic rats. MATERIALS AND METHODS: Male Wistar rats underwent HFD feeding for 4 wk and were killed at 0, 1.5, and 6 h after lipopolysaccharide (LPS) injection. Plasma levels of adiponectin, nitric oxide, and interleukin 6; messenger RNA expression of adiponectin receptors (AdipoR1 and AdipoR2) in the liver and the skeletal muscle; blood biochemical test results; and histology of the liver were analyzed. RESULTS: HFD-fed rats had a lower survival rate (12.8% versus 85.2%) and lower plasma adiponectin levels after LPS injection (P < 0.01). Messenger RNA expression of adiponectin receptors in the liver, but not the skeletal muscle, also decreased in HFD-fed rats (P < 0.05). Tissue injury and oxidative stress in the liver and plasma inflammatory mediator levels increased, and worsened lipid metabolism abnormalities were noted. The findings indicated that HFD decreased the sensitivity of adiponectin and was associated with an increase in oxidative stress and inflammation, which finally resulted in worsened liver injury and poor survival rate after LPS injection. CONCLUSIONS: Short-term, HFD-induced, mild obesity is harmful to the septic host, reduces adiponectin sensitivity, and could be the cause of worsening pathologic conditions.

Fam20C overexpression in odontoblasts regulates dentin formation and odontoblast differentiation.

FAM20C phosphorylates secretory proteins at S-x-E/pS motifs, and previous studies of Fam20C-dificient mice revealed that FAM20C played essential roles in bone and tooth formation. Inactivation of FAM20C in mice led to hypophosphatemia that masks direct effect of FAM20C in these tissues, and consequently the direct role of FAM20C remains unknown. Our previous study reported that osteoblast/odontoblast-specific Fam20C transgenic (Fam20C-Tg) mice had normal serum phosphate levels and that osteoblastic FAM20C-mediated phosphorylation regulated bone formation and resorption. Here, we investigated the direct role of FAM20C in dentin using Fam20C-Tg mice. The tooth of Fam20C-Tg mice contained numerous highly phosphorylated proteins, including SIBLINGs, compared to that of wild-type mice. In Fam20C-Tg mice, coronal dentin volume decreased and mineral density unchanged at early age, while the volume unchanged and the mineral density elevated at maturity. In these mice, radicular dentin volume and mineral density decreased at all ages, and histologically, the radicular dentin had wider predentin and abnormal apical-side dentin with embedded cells and argyrophilic canaliculi. Immunohistochemical analyses revealed that abnormal apical-side dentin had bone and dentin matrix properties accompanied with osteoblast-lineage cells. Further, in Fam20C-Tg mice, DSPP content which is important for dentin formation, was reduced in dentin, especially radicular dentin, which might lead to defects mainly in radicular dentin. Renal subcapsular transplantations of tooth germ revealed that newly formed radicular dentin replicated apical abnormal dentin of Fam20C-Tg mice, corroborating that FAM20C overexpression indeed caused the abnormal dentin. Our findings indicate that odontoblastic FAM20C-mediated phosphorylation in the tooth regulates dentin formation and odontoblast differentiation.

Prediction of cervical lymph node metastasis from immunostained specimens of tongue cancer using a multilayer perceptron neural network.

BACKGROUND: Although cervical lymph node metastasis is an important prognostic factor for oral cancer, occult metastases remain undetected even by diagnostic imaging. We developed a learning model to predict lymph node metastasis in resected specimens of tongue cancer by classifying the level of immunohistochemical (IHC) staining for angiogenesis- and lymphangiogenesis-related proteins using a multilayer perceptron neural network (MNN). METHODS: We obtained a dataset of 76 patients with squamous cell carcinoma of the tongue who had undergone primary tumor resection. All 76 specimens were IHC stained for the six types shown above (VEGF-C, VEGF-D, NRP1, NRP2, CCR7, and SEMA3E) and 456 slides were prepared. We scored the staining levels visually on all slides. We created virtual slides (4560 images) and the accuracy of the MNN model was verified by comparing it with a hue-saturation (HS) histogram, which quantifies the manually determined visual information. RESULTS: The accuracy of the training model with the MNN was 98.6%, and when the training image was converted to grayscale, the accuracy decreased to 52.9%. This indicates that our MNN adequately evaluates the level of staining rather than the morphological features of the IHC images. Multivariate analysis revealed that CCR7 staining level and T classification were independent factors associated with the presence of cervical lymph node metastasis in both HS histograms and MNN. CONCLUSION: These results suggest that IHC assessment using MNN may be useful for identifying lymph node metastasis in patients with tongue cancer.

Clear Cell Squamous Cell Carcinoma of the Maxillary Gingiva Associated with PIK3CA and HRAS Mutations: Report of a Case and Literature Review.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common oral malignancy, and somatic mutations in some driver genes have been implicated in SCC development. Clear cell SCC (CCSCC) is a rare histological variant of SCC, and various clear cell neoplasms must be considered in the differential diagnosis of CCSCC in the oral cavity. Based on a limited number of CCSCC cases reported in the oral cavity, CCSCC is considered an aggressive variant of SCC with a poor prognosis; however, its genetic characteristics remain unknown. METHODS: A maxillary gingival tumor in an 89-year-old female was described and investigated using immunohistochemical staining, special staining, fluorescence in situ hybridization, and next-generation sequencing (NGS) with a custom panel of driver genes, including those associated with SCC and clear cell neoplasm development. RESULTS: Histopathological examination revealed a proliferation of atypical epithelial cells with abundant clear cytoplasm and enlarged and centrally placed round nuclei. The tumor was exophytic with deep, penetrating proliferation. The atypical clear cells were continuous with the conventional SCC cells. Immunohistochemical analysis showed that the clear cells were positive for CK AE1/AE3 and CK5/6 and nuclear-positive for p63. In contrast, the clear cells were negative for αSMA, S100, HMB45, Melan-A, CD10, and p16. p53 immunoreactivity exhibited a wild-type expression pattern. Additionally, the clear cells were positive for periodic acid-Schiff (PAS) and negative for diastase-PAS, mucicarmine, and Alcian blue. Based on these results, the diagnosis of CCSCC was confirmed. Molecular analysis of the clear cells identified PIK3CA p.E542K (c.1624G>A) and HRAS p.G12A (c.35 G>C) somatic mutations classified as oncogenic. No pathogenic variants were identified in TP53, EWSR1, AKT1, PTEN, BRAF, KRAS, NRAS, RASA1, or MAML2. CONCLUSIONS: We report a case of CCSCC of the oral cavity with PIK3CA and HRAS mutations. The identification of PIK3CA and/or HRAS mutations is rare in SCC; however, both mutations are important potential targets for antitumor therapy. A detailed analysis of gene mutations in CCSCC may lead to a better understanding of its biological behavior and an improved prognosis, as well as a differential diagnosis from other clear cell neoplasms.

Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate.

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Though nutritional status is a known regulator of long bone growth, it is largely unknown if and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2Cre ERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone, and that exogenous IGF-1 canceled this reduction and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2Cre ERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate, and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of the chondroprogenitor cells.

Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate.

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate, where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Although nutritional status is a known regulator of long bone growth, it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2CreERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.