Suppressing Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 Activity to Enhance the Effectiveness of Anti-Cancer Drugs That Induce Multipolar Mitotic Spindles.

Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.

Identification of salivary autoantibodies as biomarkers of oral cancer with immunoglobulin A enrichment combined with affinity mass spectrometry.

Globally, oral cavity squamous cell carcinoma (OSCC) is one of the most common fatal illnesses. Its high mortality is ascribed to the fact that the disease is often diagnosed at a late stage, which indicates an urgent need for approaches for the early detection of OSCC. The use of salivary autoantibodies (autoAbs) as OSCC biomarkers has numerous advantages such as easy access to saliva samples and efficient detection of autoAbs using well-established secondary reagents. To improve OSCC screening, we identified OSCC-associated autoAbs with the enrichment of salivary autoAbs combined with affinity mass spectrometry (MS). The salivary IgA of healthy individuals and OSCC patients was purified with peptide M-conjugated beads and then applied to immunoprecipitated antigens (Ags) in OSCC cells. Using tandem MS analysis and spectral counting-based quantitation, the level of 10 Ags increased in the OSCC group compared with the control group. Moreover, salivary levels of autoAbs to the 10 Ags were determined by a multiplexed bead-based immunoassay. Among them, seven were significantly higher in early-stage OSCC patients than in healthy individuals. A marker panel consisting of autoAbs to LMAN2, PTGR1, RAB13, and UQCRC2 was further developed to improve the early diagnosis of OSCC.

MFI2 upregulation promotes malignant progression through EGF/FAK signaling in oral cavity squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the predominant histological type of the head and neck squamous cell carcinoma (HNSCC). By comparing the differentially expressed genes (DEGs) in OSCC-TCGA patients with copy number variations (CNVs) that we identify in OSCC-OncoScan dataset, we herein identified 37 dysregulated candidate genes. Among these potential candidate genes, 26 have been previously reported as dysregulated proteins or genes in HNSCC. Among 11 novel candidates, the overall survival analysis revealed that melanotransferrin (MFI2) is the most significant prognostic molecular in OSCC-TCGA patients. Another independent Taiwanese cohort confirmed that higher MFI2 transcript levels were significantly associated with poor prognosis. Mechanistically, we found that knockdown of MFI2 reduced cell viability, migration and invasion via modulating EGF/FAK signaling in OSCC cells. Collectively, our results support a mechanistic understanding of a novel role for MFI2 in promoting cell invasiveness in OSCC.

Clinical Outcomes of Taiwanese Patients with Resected Oral Cavity Squamous Cell Carcinoma Who Underwent Reconstruction with Free Versus Local Flaps.

BACKGROUND: We sought to compare the clinical outcomes of Taiwanese patients with resected oral cavity squamous cell carcinoma (OCSCC) who underwent reconstruction with free versus local flaps. METHODS: From 2011 to 2017, we examined 8646 patients with first primary OCSCC who received surgery either with or without adjuvant therapy. Of these patients, 7297 and 1349 received free and local flap reconstruction, respectively. Two propensity score-matched groups of patients who underwent free versus local flap (n = 1268 each) reconstructions were examined. Margin status was not included as a propensity score-matched variable. RESULTS: Compared with local flaps, patients who received free flaps had a higher prevalence of the following variables: male sex, age < 65 years, pT3-4, pN1-3, p-Stage III-IV, depth ≥ 10 mm, margin > 4 mm, extranodal extension (ENE), and adjuvant therapy (all p < 0.0001). Multivariable analysis identified the reconstruction method (local vs. free flaps, only overall survival [OS]), age ≥ 65 years, pT3-4, pN1-3, p-Stage III-IV, depth ≥ 10 mm (only OS), margins ≤ 4 mm, and ENE as independent adverse prognosticators for disease-specific survival (DSS) and OS. The results of propensity score-matched analyses revealed that, compared with free flaps, patients who underwent local flap reconstruction showed less favorable 5-year DSS (hazard ratio [HR] 1.26, 82%/77%; p = 0.0100) and OS (HR 1.21, 73%/68%; p = 0.0079). CONCLUSIONS: After adjusting for covariates using multivariate models, and also by propensity score modeling, OCSCC patients who underwent free flap reconstruction showed a higher frequency of clear margins and a significant survival advantage compared with those who received local flaps.

Identification of Salivary Biomarkers for Oral Cancer Detection with Untargeted and Targeted Quantitative Proteomics Approaches.

Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. In Taiwan, OSCC is the fifth leading cause of cancer-related mortality and leads to 2800 deaths per year. The poor outcome of OSCC patients is principally ascribed to the fact that this disease is often advanced at the time of diagnosis, suggesting that early detection of OSCC is urgently needed. Analysis of cancer-related body fluids is one promising approach to identify biomarker candidates of cancers. To identify OSCC biomarkers, salivary proteomes of OSCC patients, individuals with oral potentially malignant disorders (OPMDs), and healthy volunteers were comparatively profiled with isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry (MS). The salivary levels of 67 and 18 proteins in the OSCC group are elevated and decreased compared with that in the noncancerous group (OPMD and healthy groups), respectively. The candidate biomarkers were further selected using the multiple reaction monitoring (MRM)-MS and validated with the immunoassays. More importantly, the higher salivary level of three proteins, complement factor H (CFH), fibrinogen alpha chain (FGA), and alpha-1-antitrypsin (SERPINA1) was correlated with advanced stages of OSCC. Our results indicate that analysis of salivary proteome is a feasible strategy for biomarker discovery, and the three proteins are potential salivary markers for OSCC diagnosis.

Proteomic Profiling of Paired Interstitial Fluids Reveals Dysregulated Pathways and Salivary NID1 as a Biomarker of Oral Cavity Squamous Cell Carcinoma.

Patients with oral cavity squamous cell carcinoma (OSCC) are frequently first diagnosed at an advanced stage, leading to poor prognosis and high mortality rates. Early detection of OSCC using body fluid-accessible biomarkers may improve the prognosis and survival rate of OSCC patients. As tumor interstitial fluid is a proximal fluid enriched with cancer-related proteins, it is a useful reservoir suitable for the discovery of cancer biomarkers and dysregulated biological pathways in tumor microenvironments. Thus, paired interstitial fluids of tumor (TIF) and adjacent noncancerous (NIF) tissues from 10 OSCC patients were harvested and analyzed using one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Using label-free spectral counting-based quantification, 113 proteins were found to be up-regulated in the TIFs compared with the NIFs. The gene set enrichment analysis (GSEA) revealed that the differentially expressed TIF proteins were highly associated with aminoacyl tRNA biosynthesis pathway. The elevated levels of 4 proteins (IARS, KARS, WARS, and YARS) involved in the aminoacyl tRNA biosynthesis were verified in the OSCC tissues with immunohistochemistry (IHC). In addition, nidogen-1 (NID1) was selected for verification as an OSCC biomarker. Salivary level of NID1 in OSCC patients (n = 48) was significantly higher than that in the healthy individuals (n = 51) and subjects with oral potentially malignant disorder (OPMD; n = 53). IHC analysis showed that NID1 level in OSCC tissues was increased compared with adjacent noncancerous epithelium (n = 222). Importantly, the elevated NID1 level was correlated with the advanced stages of OSCC, as well as the poor survival of OSCC patients. Collectively, the results suggested that TIF analysis facilitates understanding of the OSCC microenvironment and that salivary NID1 may be a useful biomarker for OSCC.

The role of postoperative radiotherapy in pN1 oral cavity cancer without extranodal extension.

BACKGROUND: The administration of postoperative radiotherapy remains controversial in pN1 oral cavity cancer patients without extranodal extension. The aim is to determine whether postoperative radiotherapy reduces the neck recurrence rate and improves the survival outcomes of pN1 patients. METHODS: This study consecutively enrolled 1056 patients with newly diagnosed oral squamous cell carcinoma who underwent tumor wide excision and neck dissection from September 2002 to November 2019. One hundred two pN1 patients without extranodal extension were eligible for analysis. Then, a subgroup analysis of 40 patients was performed after patients with other adverse risk factors (positive margins, close margins, lymphovascular invasion, perineural invasion, tumor depth ≥ 10 mm, and poor histological differentiation) were excluded. RESULTS: Of the 102 eligible pN1 patients, 26 patients received surgery alone, and 76 received postoperative radiotherapy. No significant differences were observed in the neck recurrence rate (7.7% vs. 15.8%, p = 0.30). Similarly, in patients without other adverse risk factors, no significant differences were observed in the neck recurrence rate (5% vs. 20%, p = 0.15) between surgery alone group and postoperative radiotherapy group. Moreover, no significant difference was found in the neck recurrence-free survival rate, overall survival, and disease-specific survival (77.1% vs. 52.5%, p = 0.42, 83.5% vs. 64.5%, p = 0.81, and 88.2% vs. 67.9%, p = 0.34, respectively). CONCLUSION: Postoperative radiotherapy did not significantly decrease the probability of neck recurrence and survival outcomes in pN1 patients without extranodal extension. Radical surgery alone may be considered sufficient treatment for pN1 patients without other adverse risk factors.

A combined analysis of maximum standardized uptake value on FDG-PET, genetic markers, and clinicopathological risk factors in the prognostic stratification of patients with resected oral cavity squamous cell carcinoma.

OBJECTIVE: Clinical outcomes of patients with resected oral cavity squamous cell carcinoma (OCSCC) chiefly depend on the presence of specific clinicopathological risk factors (RFs). Here, we performed a combined analysis of FDG-PET, genetic markers, and clinicopathological RFs in an effort to improve prognostic stratification. METHODS: We retrospectively reviewed the clinical records of 2036 consecutive patients with first primary OCSCC who underwent surgery between 1996 and 2016. Of them, 345 underwent ultra-deep targeted sequencing (UDTS, between 1996 and 2011) and 168 whole exome sequencing (WES, between 2007 and 2016). Preoperative FDG-PET imaging was performed in 1135 patients from 2001 to 2016. Complete data on FDG-PET, genetic markers, and clinicopathological RFs were available for 327 patients. RESULTS: Using log-ranked tests based on 5-year disease-free survival (DFS), the optimal cutoff points for maximum standardized uptake values (SUV-max) of the primary tumor and neck metastatic nodes were 22.8 and 9.7, respectively. The 5-year DFS rates were as follows: SUVtumor-max ≥ 22.8 or SUVnodal-max ≥ 9.7 (n = 77) versus SUVtumor-max < 22.8 and SUVnodal-max < 9.7 (n = 250), 32%/62%, P < 0.001; positive UDTS or WES gene panel (n = 64) versus negative (n = 263), 25%/62%, P < 0.001; pN3b (n = 165) versus pN1-2 (n = 162), 42%/68%, P < 0.001. On multivariate analyses, SUVtumor-max ≥ 22.8 or SUVnodal-max ≥ 9.7, a positive UDTS/WES gene panel, and pN3b disease were identified as independent prognosticators for 5-year outcomes. Based on these variables, we devised a scoring system that identified four distinct prognostic groups. The 5-year rates for patients with a score from 0 to 3 were as follows: loco-regional control, 80%/67%/47%/24% (P < 0.001); distant metastases, 13%/23%/55%/92% (P < 0.001); DFS, 74%/58%/28%/7% (P < 0.001); and disease-specific survival, 80%/64%/35%/7% (P < 0.001) respectively. CONCLUSIONS: The combined assessment of tumor and nodal SUV-max, genetic markers, and pathological node status may refine the prognostic stratification of OCSCC patients.

A propositional AI system for supporting epilepsy diagnosis based on the 2017 epilepsy classification: Illustrated by Dravet syndrome.

PURPOSE: The 2017 epilepsy and seizure diagnosis framework emphasizes epilepsy syndromes and the etiology-based approach. We developed a propositional artificial intelligence (AI) system based on the above concepts to support physicians in the diagnosis of epilepsy. METHODS: We analyzed and built ontology knowledge for the classification of seizure patterns, epilepsy, epilepsy syndrome, and etiologies. Protégé ontology tool was applied in this study. In order to enable the system to be close to the inferential thinking of clinical experts, we classified and constructed knowledge of other epilepsy-related knowledge, including comorbidities, epilepsy imitators, epilepsy descriptors, characteristic electroencephalography (EEG) findings, treatments, etc. We used the Ontology Web Language with Description Logic (OWL-DL) and Semantic Web Rule Language (SWRL) to design rules for expressing the relationship between these ontologies. RESULTS: Dravet syndrome was taken as an illustration for epilepsy syndromes implementation. We designed an interface for the physician to enter the various characteristics of the patients. Clinical data of an 18-year-old boy with epilepsy was applied to the AI system. Through SWRL and reasoning engine Drool's execution, we successfully demonstrate the process of differential diagnosis. CONCLUSION: We developed a propositional AI system by using the OWL-DL/SWRL approach to deal with the complexity of current epilepsy diagnosis. The experience of this system, centered on the clinical epilepsy syndromes, paves a path to construct an AI system for further complicated epilepsy diagnosis.

The impact of preoperative glycated hemoglobin levels on outcomes in oral squamous cell carcinoma.

OBJECTIVES: This study aimed to investigate the association between preoperative glycated hemoglobin (HbA1c) levels and the treatment outcomes of oral cavity squamous cell carcinoma (OSCC). METHODS: Three hundred and fifty-eight OSCC patients were consecutively enrolled between July 2004 and July 2016. Clinicopathological parameters and survival outcomes were analyzed following HbA1c stratification of 6.5% (HbA1c ≥ 6.5%: n = 74, 20.6%) and 7.0% (HbA1c ≥ 7.0%: n = 53, 14.8%). RESULTS: Higher HbA1c levels were associated with elevated body mass index, lower albumin levels, wider surgical margins, and prolonged hospital stays (HbA1c 6.5%: p = .001, .048, .030, .009, respectively; HbA1c 7.0%: p = .092, .032, .009, .015, respectively). Survival rates stratified by HbA1c 6.5% were as follows: locoregional recurrence-free survival, p = .014; distant metastasis-free survival, p = .013; second primary cancer-free survival, p = .015; overall survival, p = .014; disease-specific survival, p = .002 and HbA1c 7.0%: locoregional recurrence-free survival, p = .013; distant metastasis-free survival, p = .013; second primary cancer-free survival, p = .014; overall survival, p = .015; disease-specific survival, p = .004. Multivariate analyses identified HbA1c as an independent prognostic factor for overall and disease-specific survival (HbA1c 6.5%: p = .014 and .002, respectively; HbA1c 7.0%: p = .036 and .013, respectively). CONCLUSIONS: Oral squamous cell carcinoma patients with higher preoperative HbA1c levels had longer hospitalization and worse survival outcomes.

Influence of Hyperglycemia on Treatment Outcomes of Oral Cavity Squamous Cell Carcinoma.

PURPOSE: The present study investigated the association between perioperative hyperglycemia and the treatment and survival outcomes of patients with oral cavity squamous cell carcinoma (OSCC). PATIENTS AND METHODS: From 2004 to 2016, 385 patients with OSCC were enrolled and stratified into normoglycemic (<180 mg/dL) and hyperglycemic (≥180 mg/dL) groups. The clinicopathologic characteristics and treatment outcomes of OSCC were subsequently analyzed. RESULTS: Of the 385 patients, 61 (15.8%) were in the hyperglycemic group. Hyperglycemia was significantly associated with pT stage, pN stage, overall pathologic stage, extranodal extension, albumin level, and tumor depth (P = .004, P = .042, P = .008, P = .001, P = .004, and P = .011, respectively). Patients with hyperglycemia also required a longer hospital stay (P = .003). The 5-year overall survival and disease-specific survival were poorer in the hyperglycemic group than in the normoglycemic group (P = .001 and P = .002, respectively). Multivariate analysis revealed that hyperglycemia is a significant adverse prognostic indicator for OSCC (hazard ratio, 1.709; 95% confidence interval, 1.003 to 2.912; P = .049). CONCLUSIONS: Hyperglycemia is associated with more advanced disease and poorer survival rates in patients with OSCC. It correlates with adverse clinicopathologic characteristics and longer hospital stay. Screening for hyperglycemia and maintenance of normal glycemic status during the treatment course is imperative in the treatment of OSCC.

Treatment patterns and survival outcomes of advanced hypopharyngeal squamous cell carcinoma.

BACKGROUND: This study evaluated the treatment outcomes of the primary surgery (PS) or concurrent chemoradiotherapy (CCRT) as the initial treatment for hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: This retrospective cohort study included patients with stages III-IV HPSCC from four tertiary referral centers consecutively enrolled from 2003 to 2012; of them, 213 (32.6%) and 439 (67.4%) had received PS and CCRT as their primary treatments, respectively. The 5-year overall survival (OS) and disease-free survival (DFS) rates were analyzed using the Kaplan-Meier method and Cox regression models. RESULTS: In patients undergoing PS and CCRT, OS rates were 45.0% and 33.1% (p < 0.001), respectively, and DFS rates were 36.2% and 28.9% (p = 0.003), respectively. In subgroup analysis, in patients with stage IVA HPSCC, PS was associated with higher OS rate (p = 0.002), particularly in those with T4 or N2 classification (p = 0.021 and 0.002, respectively). Multivariate analysis indicated that stage IVA HPSCC, stage IVB HPSCC, and CCRT were independent adverse prognostic factors for OS rate (p = 0.004, < 0.001, and 0.014, respectively). Furthermore, in patients with stage IVA HPSCC aged ≥ 65 years and with N2 classification, CCRT was significantly associated with lower OS rates than was PS (p = 0.027 and 0.010, respectively). CONCLUSIONS: In patients with advanced HPSCC, PS was significantly associated with better prognosis than CCRT. PS could be a favorable primary treatment modality for the management of patients with stage IVA HPSCC, particularly those aged ≥ 65 years and with T4 and N2 classification.

Aesthetic single-stage vermilion reconstruction using facial artery musculomucosal flap and radial forearm free flap following cancer resection: A case report.

Reconstruction of defects measuring approximately two-thirds of the lower lip width is traditionally reconstructed utilizing loco-regional flap utilizing lip and cheek tissues. This often results in microstomia and unsatisfactory aesthetic outcome. This may hinder the psychosocial aspect of a recovering cancer survivor. Here we describe a single-stage reconstruction in a 79-year-old male patient who received lower lip resection for squamous cell carcinoma. The reconstruction was accomplished using a facial artery musculomucosal flap together with a free radial forearm flap for vermilion and soft tissue defect reconstruction after lip tumor resection with uneventful postoperative course. The patient remains disease-free after 2-year follow-up and does not require revision surgeries for functional or aesthetic reason. This approach may be considered a good option for reconstruction of missing lip and soft tissue simultaneously when the facial vessels are well-preserved during neck dissection. Aesthetically pleasing and functionally satisfactory outcomes may be produced.

Anti-p53 Autoantibody Detection in Automatic Glass Capillary Immunoassay Platform for Screening of Oral Cavity Squamous Cell Carcinoma.

The incidence of oral squamous cell carcinoma (OSCC), which is one of the most common cancers worldwide, has been increasing. Serum anti-p53 autoantibody is one of the most sensitive biomarkers for OSCC. Currently, the most commonly used method on clinical screening platforms is the enzyme-linked immunosorbent assay, owing to its high specificity and repeatability. However, conducting immunoassays on 96-well plates is typically time consuming, thereby limiting its clinical applications for fast diagnosis and immediate prognosis of rapidly progressive diseases. The present study performed immunoassays in glass capillaries of 1-mm internal diameter, which increases the surface to volume ratio of the reaction, to shorten the time needed for immunoassay. The immunoassay was automated while using linear motorized stages and a syringe pump. The results indicated that, when compared with the 96-well plate immunoassay, the glass capillary immunoassay decreased the reaction time from typical 120 min to 45 min, reduced the amount of reagent from typical 50 µL to 15 µL, and required only simple equipment setup. Moreover, the limit of detection for glass capillary anti-p53 autoantibody immunoassay was 0.46 ng mL-1, which is close to the 0.19 ng mL-1 value of the conventional 96-well plate assay, and the glass capillary method had a broader detection range. The apparatus was used to detect the serum anti-p53 autoantibody concentration in clinical patients and compare its results with the conventional 96-well plate method results, which suggested that both of the methods detect the same trend in the relative concentration of serum anti-p53 autoantibody in healthy individuals or patients with OSCC.

Characterization of Copy Number Variations in Oral Cavity Squamous Cell Carcinoma Reveals a Novel Role for MLLT3 in Cell Invasiveness.

BACKGROUND: DNA copy number variations (CNVs) are a hallmark of cancer, and the current study aimed to demonstrate the profile of the CNVs for oral cavity squamous cell carcinoma (OSCC) and elucidate the clinicopathological associations and molecular mechanisms of a potential marker derived from CNVs, mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), in OSCC carcinogenesis. MATERIALS AND METHODS: CNVs in 37 OSCC tissue specimens were analyzed using a high-resolution microarray, the OncoScan array. Gene expression was analyzed by real-time polymerase chain reaction in 127 OSCC and normal tissue samples. Cell function assays included cell cycle, migration, invasion and chromatin immunoprecipitation assays. RESULTS: We found a novel copy number amplified region, chromosome 9p, encompassing MLLT3 via the comparison of our data set with six other OSCC genome-wide CNV data sets. MLLT3 overexpression was associated with poorer overall survival in patients with OSCC (p = .048). MLLT3 knockdown reduced cell migration and invasion. The reduced invasion ability in MLLT3-knockdown cells was rescued with double knockdown of MLLT3 and CBP/p300-interacting transactivator with ED rich carboxy-terminal domain 4 (CITED4; 21.0% vs. 61.5%). Knockdown of MLLT3 impaired disruptor of telomeric silencing-1-like (Dot1L)-associated hypermethylation in the promoter of the tumor suppressor, CITED4 (p < .001), and hence dysregulated HIF-1α-mediated genes (TWIST, MMP1, MMP2, VIM, and CDH1) in OSCC cells. CONCLUSION: We identified unique CNVs in tumors of Taiwanese patients with OSCC. Notably, MLLT3 overexpression is related to the poorer prognosis of patients with OSCC and is required for Dot1L-mediated transcriptional repression of CITED4, leading to dysregulation of HIF-1α-mediated genes. IMPLICATIONS FOR PRACTICE: This article reports unique copy number variations in oral cavity squamous cell carcinoma (OSCC) tumors of Taiwanese patients. Notably, MLLT3 overexpression is related to the poorer prognosis of patients with OSCC and is required for Dot1L-mediated transcriptional repression of CITED4, leading to dysregulation of HIF-1α-mediated genes.

Tumor Depth of Invasion (Tumor > 4 cm/Depth > 10 mm and Depth > 20 mm) and Through Cortex/Skin Invasion are Both Valid Criteria for Classifying Tumors as pT4a in AJCC 2018 Oral Cavity Cancer Staging System.

BACKGROUND: According to the AJCC third to seventh edition staging manuals (1988-2010), the presence of through cortex and/or skin invasion in oral cavity squamous cell carcinoma (OCSCC) identifies T4a tumors. The AJCC eighth edition (2018) introduced a depth of invasion (DOI) > 20 mm as a criterion for pT4a. Subsequently, a revision maintained that tumors > 4 cm with a DOI > 10 mm should be classified as pT4a. We sought to analyze the prognostic impact of the three distinct criteria identifying pT4a disease. METHODS: We examined 667 consecutive patients with pT3-4 buccal/gum/hard palate/retromolar SCC who underwent surgery between 1996 and 2016. pT1/pT2 (n = 108/359) disease were included for comparison purposes. RESULTS: The 5-year outcomes of patients with pT1/pT2/without (n = 406)/with tumor > 4 cm/DOI > 10 mm (n = 261), pT1/pT2/DOI ≤ 20 mm (n = 510)/> 20 mm (n = 157), and pT1/pT2/without (n = 305)/with through cortex/skin invasion (n = 362) were as follows: disease-specific survival (DSS), 98%/89%/79%/65%, p < 0.001, 98%/89%/78%/59%, p < 0.001, and 98%/89%79%/69%, p < 0.001; overall survival (OS), 90%/79%/63%/51%, p < 0.001, 90%/79%/63%/42%, p < 0.001, and 90%/79%/65%/52%, p < 0.001. In pT3-4 disease, a tumor > 4 cm/DOI > 10 mm was an independent adverse prognosticator for 5-year DSS rate, DOI > 20 mm was an independent adverse prognosticator for 5-year DSS and OS rates, whereas through cortex/skin invasion independently predicted 5-year OS rates. CONCLUSIONS: All of the three criteria (tumor > 4 cm/DOI > 10 mm, DOI > 20 mm, and through cortex/skin invasion) identify high-risk patients, which should be reflected in further revisions of pT4a classification in OCSCC.

Value of early evaluation of treatment response using 18F-FDG PET/CT parameters and the Epstein-Barr virus DNA load for prediction of outcome in patients with primary nasopharyngeal carcinoma.

PURPOSE: To determine the value of early evaluation of response to concurrent chemoradiotherapy (CCRT) using 18F-FDG PET-derived parameters and the Epstein-Barr virus (EBV) DNA titre in outcome prediction in patients with primary nasopharyngeal carcinoma (NPC). METHODS: Sixty patients with primary NPC were prospectively enrolled. All patients underwent 18F-FDG PET/CT before and during CCRT. The plasma EBV DNA titre was measured along with the PET/CT-derived parameters. Changes in EBV DNA titre and PET/CT-derived parameters during CCRT were analysed in relation to response to treatment, recurrence-free survival (RFS) and overall survival (OS). RESULTS: A total lesion glycolysis (TLG) reduction ratio of ≤0.6 and a detectable EBV DNA titre during CCRT were predictors of an unfavourable response to treatment, RFS and OS. In multivariate analysis, a TLG reduction ratio of ≤0.6 predicted incomplete remission (p = 0.002) and decreased RFS (p = 0.003). The proportion of patients with a TLG reduction ratio of >0.6 who achieved a complete response was more than twice that of patients with a TLG reduction ratio of ≤0.6. A detectable EBV DNA titre, a TLG reduction ratio of ≤0.6 and older age were independently associated with a poorer OS (p = 0.037, 0.009 and 0.016, respectively). A scoring system was developed based on these independent predictors of OS. Patients with a score of 1 and 2/3 had poorer survival outcomes than those with a score of 0 (hazard ratio 4.756, p = 0.074, and hazard ratio 18.973, p = 0.001, respectively). This scoring system appeared to be superior to the traditional TNM staging system (p < 0.001 versus p = 0.175). CONCLUSION: Early evaluation of response to CCRT using 18F-FDG PET-derived parameters and the EBV DNA titre can predict outcome in patients with primary NPC. A combination of interim PET parameters and the EBV DNA titre enables better stratification of patients into subgroups with different survival rates.

BRAF protein immunoprecipitation, elution, and digestion from cell extract using a microfluidic mixer for mutant BRAF protein quantification by mass spectrometry.

This study utilized a microfluidic mixer for the sample pretreatment of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation and protein enzymatic digestion. The time of sample pretreatment was reduced and thus the throughput of quantitative mutant proteins was increased by using the proposed method. Whole cell lysates of the cancer cell line HT-29 with gene mutations were used as the sample. The target protein BRAF was immunoprecipitated using magnetic beads in a pneumatic micromixer. Purified protein was then eluted and digested by trypsin in another two micromixers to yield peptide fragments in the solution. Using stable isotope-labeled standard as the internal control, wild-type and mutant BRAF proteins were quantified using mass spectrometry, which could be used for cancer screening. Compared with conventional methods in which protein immunoprecipitation lasts overnight, the micromixer procedure takes only 1 h, likely improving the throughput of mutant BRAF protein quantification by mass spectrometry. Graphical abstract Three micromixers were used to reduce the sample pretreatment time of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation, protein elution, and protein enzymatic digestion.

Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry.

Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. Although many potential oral cancer biomarkers have been discovered, very few have been verified in body fluid specimens in parallel to evaluate their clinical utility. The lack of appropriate multiplexed assays for chosen targets represents one of the bottlenecks to achieving this goal. In the present study, we develop a peptide immunoaffinity enrichment-coupled multiple reaction monitoring-mass spectrometry (SISCAPA-MRM) assay for verifying multiple reported oral cancer biomarkers in saliva. We successfully produced 363 clones of mouse anti-peptide monoclonal antibodies (mAbs) against 36 of 49 selected targets, and characterized useful mAbs against 24 targets in terms of their binding affinity for peptide antigens and immuno-capture ability. Comparative analyses revealed that an equilibrium dissociation constant (KD ) cut-off value < 2.82 × 10-9 m could identify most clones with an immuno-capture recovery rate >5%. Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-µg saliva matrix background. This multiplexed assay showed reasonable precision (median coefficient of variation, 7.16 to 32.09%), with lower limits of quantitation (LLOQ) of <10, 10-50, and >50 ng/ml for 14, 7 and 3 targets, respectively. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. Finally, we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. We propose that this assay could be used in future studies to compare the clinical utility of multiple oral cancer biomarker candidates in a large cohort of saliva samples.

Prevalence and demographic characteristics of comorbid epilepsy in children and adolescents with cerebral palsy: a nationwide population-based study.

INTRODUCTION: The nationwide prevalence of cerebral palsy (CP) is unknown due to the lack of a population-based registration system for CP in Taiwan. This study was the largest nationwide, population-based, cross-sectional study to estimate the prevalence of CP, prevalence rates of comorbid epilepsy in patients with CP, and association with socioeconomic status (SES) in Taiwan. The crude prevalence rate and age- and gender-specific prevalence rates were estimated. METHODS: A total of 8419 patients with CP were enrolled, and the estimated prevalence of CP was 1.76‰ in the pediatric population and 1.51‰ and 1.98‰ in girls and boys, respectively. The prevalence rate of epilepsy in patients with CP was 29.8%. RESULTS: The result revealed a higher prevalence of CP and epileptic CP in members of families with lower insurance premiums than those with higher insurance premiums and those from East Taiwan compared with those from other areas of Taiwan. Moreover, a higher prevalence of CP is shown in rural area than urban area. DISCUSSION: SES and geographic variables were significantly associated with the risk of epilepsy in children with CP. Patients with epileptic CP had a higher odds ratio of several neuropsychiatric diseases, including mental retardation, ophthalmologic problems, hearing impairment, and hydrocephalus.