Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer.

Bromodomain and extraterminal domain (BET) family proteins are epigenetic master regulators of gene expression via recognition of acetylated histones and recruitment of transcription factors and co-activators to chromatin. Hence, BET family proteins have emerged as promising therapeutic targets in cancer. In this study, we examined the functional role of bromodomain containing 3 (BRD3), a BET family protein, in colorectal cancer (CRC). In vitro and vivo analyses using BRD3-knockdown or BRD3-overexpressing CRC cells showed that BRD3 suppressed tumor growth and cell cycle G1/S transition and induced p21 expression. Clinical analysis of CRC datasets from our hospital or The Cancer Genome Atlas revealed that BET family genes, including BRD3, were overexpressed in tumor tissues. In immunohistochemical analyses, BRD3 was observed mainly in the nucleus of CRC cells. According to single-cell RNA sequencing in untreated CRC tissues, BRD3 was highly expressed in malignant epithelial cells, and cell cycle checkpoint-related pathways were enriched in the epithelial cells with high BRD3 expression. Spatial transcriptomic and single-cell RNA sequencing analyses of CRC tissues showed that BRD3 expression was positively associated with high p21 expression. Furthermore, overexpression of BRD3 combined with knockdown of, a driver gene in the BRD family, showed strong inhibition of CRC cells in vitro. In conclusion, we demonstrated a novel tumor suppressive role of BRD3 that inhibits tumor growth by cell cycle inhibition in part via induction of p21 expression. BRD3 activation might be a novel therapeutic approach for CRC.

Clinical Analysis of En Bloc Resection for Advanced Temporal Bone Squamous Cell Carcinoma.

Objective En bloc and margin-negative surgical resection seems to offer the best prognosis for patients with temporal bone squamous cell carcinoma (TB-SCC). In this study, we summarize the outcomes of surgical cases of advanced TB-SCC (T3-T4) that were managed in two institutions, with an accompanying description of the surgical procedure that was utilized: modified subtotal temporal bone resection (STBR), which involves the en bloc removal of the temporal bone including or transecting the otic capsule. Design This is a case series study with chart review. Setting The study was conducted at two academic tertiary care medical centers. Participants Chart information was collected for all patients who underwent surgical resection of advanced TB-SCC between July 1998 and February 2019. The resulting dataset contained 43 patients with advanced TB-SCC who underwent en bloc resection during the review period. Tumor staging followed the modified Pittsburgh classification. Disease-specific survival (DSS) rates were calculated according to the Kaplan-Meier method. Main Outcome Measure This study shows disease-specific 5-year DSS rate. Results The 5-year DSS rate of the cases who underwent en bloc resection was 79.7%. En bloc lateral temporal bone resection was employed in a total of 25 cases (DSS: 79.0%). En bloc modified STBR was utilized in 18 cases (DSS: 81.7%). Conclusion En bloc margin-negative resection is a reliable treatment strategy for advanced TB-SCC. Modified STBR can be a treatment option for TB-SCC without marked posterior extension.

GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein.

Colorectal cancer (CRC) is one of the most common types of cancer and a significant cause of cancer mortality worldwide. Further improvements of CRC therapeutic approaches are needed. BCL2-associated athanogene 6 (BAG6), a multifunctional scaffold protein, plays an important role in tumor progression. However, regulation of BAG6 in malignancies remains unclear. This study showed that guided entry of tail-anchored proteins factor 4 (GET4), a component of the BAG6 complex, regulates the intercellular localization of BAG6 in CRC. Furthermore, GET4 was identified as a candidate driver gene on the short arm of chromosome 7, which is often amplified in CRC, by our bioinformatics approach using the CRC dataset from The Cancer Genome Atlas. Clinicopathologic and prognostic analyses using CRC datasets showed that GET4 was overexpressed in tumor cells due to an increased DNA copy number. High GET4 expression was an independent poor prognostic factor in CRC, whereas BAG6 was mainly overexpressed in the cytoplasm of tumor cells without gene alteration. The biological significance of GET4 was examined using GET4 KO CRC cells generated with CRISPR-Cas9 technology or transfected CRC cells. In vitro and in vivo analyses showed that GET4 promoted tumor growth. It appears to facilitate cell cycle progression by cytoplasmic enrichment of BAG6-mediated p53 acetylation followed by reduced p21 expression. In conclusion, we showed that GET4 is a novel driver gene and a prognostic biomarker that promotes CRC progression by inducing the cytoplasmic transport of BAG6. GET4 could be a promising therapeutic molecular target in CRC.

Oxysterol binding protein-like 3 (OSBPL3) is a novel driver gene that promotes tumor growth in part through R-Ras/Akt signaling in gastric cancer.

Gastric cancer (GC) is one of the most lethal malignant tumors. To improve the prognosis of GC, the identification of novel driver genes as therapeutic targets is in urgent need. Here, we aimed to identify novel driver genes and clarify their roles in gastric cancer. OSBPL3 was identified as a candidate driver gene by in silico analysis of public genomic datasets. OSBPL3 expression was analyzed by RT-qPCR and immunohistochemistry in GC cells and tissues. The biological functions and mechanisms of OSBPL3 in GC were examined in vitro and in vivo using GC cells. The association between OSBPL3 expression and clinical outcome in GC patients was also evaluated. Overexpression of OSBPL3 was detected in GC cells with OSBPL3 DNA copy number gains and promoter hypomethylation. OSBPL3-knockdown reduced GC cell growth in vitro and in vivo by inhibiting cell cycle progression. Moreover, an active Ras pull-down assay and western blotting demonstrated that OSBPL3 activates the R-Ras/Akt signaling pathway in GC cells. In a clinical analysis of two GC datasets, high OSBPL3 expression was predictive of a poor prognosis. Our findings suggest that OSBPL3 is a novel driver gene stimulating the R-Ras/Akt signaling pathway and a potential therapeutic target in GC patients.

Management of Residual Hearing with Cartilage Conduction Hearing Aid after Lateral Temporal Bone Resection: Our Institutional Experience.

BACKGROUND: There is no guideline for hearing compensation after temporal bone resection. This study aimed to retrospectively analyze surgical cases with reconstruction for hearing preservation after temporal bone malignancy resection and propose a new alternative to compensate for hearing loss. METHODS: We retrospectively reviewed the medical records of 30 patients who underwent lateral temporal bone surgery for temporal bone malignancy at our institution and examined their hearing abilities after surgery. RESULT: The hearing outcomes of patients with an external auditory meatus reconstruction varied widely. The mean postoperative air-bone gap at 0.5, 1, 2, and 4 kHz ranged from 22.5 dB to 71.25 dB. On the other hand, the average difference between the aided sound field thresholds with cartilage conduction hearing aid and bone conduction thresholds at 0.5, 1, 2, and 4 kHz ranged from -3.75 to 41.25. More closely located auricular cartilage and temporal bone resulted in smaller differences between the aided sound field and bone conduction thresholds. CONCLUSIONS: There is still room for improvement of surgical techniques for reconstruction of the auditory meatus to preserve hearing after temporal bone resection. The cartilage conduction hearing aid may provide non-invasive postoperative hearing compensation after lateral temporal bone resection.

Programmed Death-Ligand 1 Expression and Tumor-Infiltrating Lymphocytes in Temporal Bone Squamous Cell Carcinoma.

OBJECTIVES/HYPOTHESIS: The tumor immune microenvironment in temporal bone squamous cell carcinoma (TBSCC), including the programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs), has not been established. STUDY DESIGN: Retrospective cohort study. METHODS: We performed immunohistochemistry analyses to retrospectively analyze 123 TBSCC cases for PD-L1 expression and TILs and their prognostic significance. We also evaluated the prognostic correlations between these immunomarkers and the therapeutic responses to chemoradiotherapy (CRT). RESULTS: PD-L1 expression (≥1%) was detected in 62 (50.4%) TBSCC cases and was significantly associated with worse prognosis: progression-free survival (PFS), P < .0001; overall survival (OS), P = .0009. A high density of CD8+ TILs was significantly associated with better prognosis (PFS, P = .0012; OS, P = .0120). In contrast, a high density of Foxp3+ TILs tended to be associated with an unfavorable prognosis (PFS, P = .0148; OS, P = .0850). With regard to the tumor microenvironment subtypes defined by CD8+ TILs and PD-L1 expression, the CD8low /PD-L1+ group showed significantly worse prognosis. Among the 36 neoadjuvant CRT-treated cases, PD-L1 expression was significantly associated with worse OS (P = .0132). Among the 32 CRT-treated cases without surgery, a high density of CD8+ TILs tended to be more highly associated with complete response to CRT compared to a low density of CD8+ TILs (P = .0702). CONCLUSIONS: These results indicate that the evaluation of the tumor immune microenvironment may contribute to the prediction of prognoses and the selection of an individualized therapeutic strategy for patients with TBSCC. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2674-2683, 2021.

Establishment and characterization of a primary cell culture derived from external auditory canal squamous cell carcinoma.

There are no human cancer cell lines of external auditory canal origin available for research use. This report describes the establishment of a culture condition for external auditory canal squamous cell carcinoma, derived from human tumor tissue. Successive squamous cell carcinoma colonies were dissociated by trypsin, subcultured, and maintained on a feeder layer (MMC-TIG-1-20), yielding a clonally proliferating cell culture. Two morphological types of colony were observed: (a) densely packed colonies and (b) colonies with indistinct boundaries characterized by cell-cell complexes with fibroblast feeder cells. The SCC-like characteristics of these cells were evidenced by positivity for p53, SCCA1/2, cytokeratin, and vimentin, and cancer stem cell properties were indicated by positivity for CD44, CD133, Oct3/4, and alkaline phosphatase (ALP). One of the unique properties of cell cultures is their tendency to form steric colonies in vitro on feeder layer cells. In addition, in the presence of fresh macrophages, the cells very slowly transform to break away from colonies as free cells, a process that resembles the epidermal-mesenchymal transition, whereby cell-cell interactions are weakened and migration activity is enhanced. These factors are purported to play a key role in cancer cell metastasis.

Prognostic Significance of Systemic Inflammatory Response in Cases of Temporal Bone Squamous Cell Carcinoma.

OBJECTIVE/HYPOTHESIS: Squamous cell carcinoma (SCC) of the temporal bone is an extremely rare condition. This rarity has led to a delay in the establishment of a standard treatment protocol and adequate staging system. Identification of prognostic markers of this disease from a variety of fields is desirable in the establishment of treatment guidelines for temporal bone SCC. The aim of this study is to assess the prognostic role of inflammation-based prognostic scores in cases of temporal bone SCC. STUDY DESIGN: Case reries with chart review. METHODS: A total of 71 cases of primary malignancy eligible for curative treatment at a single tertiary medical institute were retrospectively analyzed. Univariate and multivariate regression analyzes were used to investigate the association between the inflammation-based scores and 5-year overall survival. RESULTS: Univariate Cox regression analyzes showed that a high neutrophil-to-lymphocyte ratio, high platelet-to-lymphocyte ratio, low lymphocyte-to-monocyte ratio, a Glasgow prognostic score of 2, and the systemic inflammation score of 2 were significantly associated with a poor prognosis, as well as a classification of T4 stage, presence of cervical lymph node metastasis, high white blood cell counts, and high C-reactive protein levels. The multivariate analysis showed that a clinical stage of T4 and a systemic inflammation score of 2 were independent prognostic markers. CONCLUSIONS: Inflammation-based prognostic markers are associated with the survival of patients with temporal bone SCC, as well as other head and neck SCCs. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:1782-1789, 2021.

The novel driver gene ASAP2 is a potential druggable target in pancreatic cancer.

Targeting mutated oncogenes is an effective approach for treating cancer. The 4 main driver genes of pancreatic ductal adenocarcinoma (PDAC) are KRAS, TP53, CDKN2A, and SMAD4, collectively called the "big 4" of PDAC, however they remain challenging therapeutic targets. In this study, ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 (ASAP2), one of the ArfGAP family, was identified as a novel driver gene in PDAC. Clinical analysis with PDAC datasets showed that ASAP2 was overexpressed in PDAC cells based on increased DNA copy numbers, and high ASAP2 expression contributed to a poor prognosis in PDAC. The biological roles of ASAP2 were investigated using ASAP2-knockout PDAC cells generated with CRISPR-Cas9 technology or transfected PDAC cells. In vitro and in vivo analyses showed that ASAP2 promoted tumor growth by facilitating cell cycle progression through phosphorylation of epidermal growth factor receptor (EGFR). A repositioned drug targeting the ASAP2 pathway was identified using a bioinformatics approach. The gene perturbation correlation method showed that niclosamide, an antiparasitic drug, suppressed PDAC growth by inhibition of ASAP2 expression. These data show that ASAP2 is a novel druggable driver gene that activates the EGFR signaling pathway. Furthermore, niclosamide was identified as a repositioned therapeutic agent for PDAC possibly targeting ASAP2.

Primary Advanced Squamous Cell Carcinoma of the Temporal Bone: A Single-Center Clinical Study.

OBJECTIVES/HYPOTHESIS: The extreme rarity of temporal bone squamous cell carcinoma (TB-SCC) has delayed the accumulation of high-quality clinical evidence. For the purposes of retrospective meta-analysis in the future, a large dataset with information from various institutions would be ideal. Our objective here was to retrospectively review cases of TB-SCC encountered at a single tertiary referral center and explore survival outcomes and prognostic factors. STUDY DESIGN: Retrospective chart review. METHODS: The medical records of all TB-SCC cases were retrospectively reviewed. The resulting dataset contained 71 cases of primary cancer eligible for initial definitive (curative) treatment. RESULTS: T4 status was associated with lower disease-specific 5-year survival than T1 to T3 staging (T1: 100%, T2: 92%, T3: 86%, T4: 51%). Survival was significantly higher in operable than in inoperable cases, even when restricted to advanced (T3/T4) cancers. The tumor extension to the middle ear cavity was observed in 13/17 of T3 cases, but it was not associated with poor survival. In addition, among operable cases, negative surgical margins were associated with significantly higher survival than positive margins. CONCLUSIONS: Definitive treatments can offer disease-specific 5-year survival of over 85% in T1 to T3 cases of TB-SCC. The tumor extension to the middle ear cavity is not associated with poor survival. T4 status, inoperability, nodal invasion, and positive surgical margin are identified as a predictor of poor prognosis. Still, the matter of how to deal with unresectable tumors remains an outstanding issue in the treatment of TB-SCC. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E583-E589, 2021.

Prognostic Impact of Tumor Extension in Patients With Advanced Temporal Bone Squamous Cell Carcinoma.

Objective: The extreme rarity of temporal bone squamous cell carcinoma (TB-SCC) has delayed the accumulation of high-quality clinical evidence. Our objective here was to explore anatomical factors associated with the prognosis of T4 TB-SCC cases. Study Design: Case series with chart review. Setting: Two academic tertiary care medical centers. Subjects and Methods: The medical records of all TB-SCC cases were retrospectively reviewed in two institutions. The resulting data set contained 30 cases of primary T4 cancer eligible for initial definitive (curative) treatment. Disease-specific survival was calculated according to the Kaplan-Meier method. Cox proportional hazards model was used to identify anatomical prognosis factors. Results: The disease-specific 5-years survival rate of 30 cases of T4 TB-SCC was 53.9%. The tumor invasion to the pterygoid muscle, posterior fossa dura, and sigmoid sinus and destruction of the ossicles were associated with poor prognosis in univariate analysis. The multivariate analysis reveals that the invasion of the ossicles, posterior fossa dura, and sigmoid sinus is an independent prognostic factor [hazard ratio (HR): 4.528 (95% CI: 1.161-17.658), p = 0.030; HR: 5.135 (95% CI: 1.616-16.315), p = 0.006; HR: 4.292 (95% CI: 1.385-13.303), p = 0.012]. The invasion of the carotid canal, petrous apex, middle fossa dura, otic capsule, pterygoid muscle, and middle ear had a high HR (HR > 2). The more invaded anatomical factors present in patients resulted in a poorer patient disease-specific prognosis, with a statistically significant difference. Conclusions: Assessing which anatomical structures are susceptible to invasion by tumors may be important for predicting TB-SCC patient prognosis and selecting appropriate treatment planning, especially surgical intervention. In addition to previously reported factors, the destruction of the ossicles in the middle ear cavity can be an anatomical prognosis factor.

Genetic landscape of external auditory canal squamous cell carcinoma.

External auditory canal squamous cell carcinoma (EACSCC) is an extremely rare and aggressive malignancy. Due to its rarity, the molecular and genetic characteristics of EACSCC have not yet been elucidated. To reveal the genetic alterations of EACSCC, we performed whole exome sequencing (WES) on 11 primary tumors, 1 relapsed tumor and 10 noncancerous tissues from 10 patients with EACSCC, including 1 with a rare case of synchronous bilateral EACSCC of both ears. WES of the primary tumor samples showed that the most frequently mutated gene is TP53 (63.6%). In addition, recurrent mutations in CDKN2A, NOTCH1, NOTCH2, FAT1 and FAT3 were detected in multiple samples. The mutational signature analysis of primary tumors indicated that the mutational processes associated with the activation of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases are the most common in EACSCC, suggesting its similarity to SCC from other primary sites. Analysis of arm-level copy number alterations detected notable amplification of chromosomes 3q, 5p and 8q as well as deletion of 3p across multiple samples. Focal chromosomal aberrations included amplifications of 5p15.33 (ZDHHC11B) and 7p14.1 (TARP) as well as deletion of 9p21.3 (CDKN2A/B). The protein expression levels of ZDHHC11B and TARP in EACSCC tissues were validated by immunohistochemistry. Moreover, WES of the primary and relapsed tumors from a case of synchronous bilateral EACSCC showed the intrapatient genetic heterogeneity of EACSCC. In summary, this study provides the first evidence for genetic alterations of EACSCC. Our findings suggest that EACSCC mostly resembles other SCC.

Therapeutic effect of Nivolumab for advanced / recurrent temporal bone squamous cell carcinoma.

OBJECTIVE: The immune checkpoint inhibitor Nivolumab was approved for the treatment of platinum-refractory head and neck squamous cell carcinoma (SCC), expanding the treatment options for recurrent or advanced head and neck SCC. However, since temporal bone squamous cell carcinoma (TB-SCC) is very rare cancer, the effectiveness of Nivolumab remains unclear. We investigated the effects of Nivolumab for TB-SCC. METHOD: Chart information was collected for all patients who underwent the first administration of Nivolumab for recurrent or residual TB-SCC in our hospital between September 2017 and December 2019. Tumor staging followed the modified Pittsburgh classification. Changes in the tumor burden and survival outcome were examined. RESULTS: We examined 9 patients with recurrent or residual TB-SCC who started administration of Nivolumab. In these cases, recurrent or residual SCC was observed after chemotherapy and/or chemoradiotherapy including platinum. The duration of Nivolumab was 2-54 weeks (median 20.0 weeks). The evaluation of the therapeutic effect according to the RECIST method showed partial response in 1 case, stable disease in 2 cases, progressive disease in 4 cases, and size unevaluated in 2 case. Although the number of cases was small, comparing with 5 cases without Nivolumab, these cases showed longer overall survival (1-year OS 33.3% vs 20.0%). CONCLUSION: We used Nivolumab as palliative chemotherapy in 9 patients with recurrent/residual TB-SCC, and we were able to obtain a certain therapeutic effect on TB-SCC as well as other head and neck SCC.

GTF2IRD1 on chromosome 7 is a novel oncogene regulating the tumor-suppressor gene TGFßR2 in colorectal cancer.

Chromosome 7q (Ch.7q) is clonally amplified in colorectal cancer (CRC). We aimed to identify oncogenes on Ch.7q that are overexpressed through DNA copy number amplification and determine the biological and clinical significance of these oncogenes in CRC. We identified general transcription factor 2I repeat domain-containing protein 1 (GTF2IRD1) as a potential oncogene using a CRC dataset from The Cancer Genome Atlas with a bioinformatics approach. We measured the expression of GTF2IRD1 in 98 patients with CRC using immunohistochemistry and RT-quantitative PCR (RT-qPCR). The biological effects of GTF2IRD1 expression were explored by gene set enrichment analysis (GSEA). Next, we undertook in vitro cell proliferation and cell cycle assays using siGTF2IRD1-transfected CRC cells. We further investigated the oncogenic mechanisms through which GTF2IRD1 promoted CRC progression. Finally, we assessed the clinical significance of GTF2IRD1 expression by RT-qPCR. GTF2IRD1 was overexpressed in tumor cells and liver metastatic lesions. The GSEA revealed a positive correlation between GTF2IRD1 expression and cell cycle progression-related genes. GTF2IRD1 knockdown inhibited cell proliferation and induced cell cycle arrest in Smad4-mutated CRC. GTF2IRD1 downregulated the expression of the gene encoding transforming growth factor ß receptor 2 (TGFßR2), a tumor-suppressor gene in Smad4-mutated CRC. On multivariate analysis, high GTF2IRD1 expression was an independent poor prognostic factor. Clinicopathological analysis showed that GTF2IRD1 expression was positively correlated with liver metastasis. In conclusion, GTF2IRD1 promoted CRC progression by downregulating TGFßR2 and could be a prognostic biomarker on Ch.7q in CRC. GTF2IRD1 could also be a novel oncogene in CRC.