[Construction of a prediction model for predicting rebleeding in patients with obscure gastrointestinal bleeding after capsule endoscopy].

Objective: To explore the risk factors of rebleeding in patients with obscure gastrointestinal bleeding (OGIB) after capsule endoscopy (CE), and construct a model to predict rebleeding. Methods: The data of patients with OGIB who underwent CE in Zhongda Hospital Affiliated to Southeast University from July 2018 to September 2021 were retrospectively analyzed. Follow-up data were obtained via electronic medical records or telephone interviews. Univariate and multivariate Cox regression models were performed to figure out the risk factors of rebleeding in OGIB patients. Then the optimal prediction model was determined and presented as a nomogram. The model was evaluated by C statistic, calibration curve and decision curve analysis. Results: One hundred and thirty patients with OGIB were included, including 64 females and 66 males, aged (55.8±17.2) years (18-87 years), and 39 (30.0%) cases developed rebleeding during follow-up. Univariate and multivariate Cox regression model analysis showed the duration of more than 2 weeks OGIB (HR=3.70, 95%CI: 1.85-7.42, P<0.001), a history of previous gastrointestinal bleeding (HR=5.25, 95%CI: 2.00-13.81, P<0.001), positive CE findings (HR=3.72, 95%CI: 1.66-8.33, P=0.001), and the lowest hemoglobin level before CE<80 g/L (HR=2.00, 95%CI: 1.02-3.84, P=0.044) were risk factors for rebleeding, while specific treatment (HR=0.25, 95%CI: 0.11-0.54, P<0.001) was a protective factor. The corresponding scores of the above five predictive factors were: OGIB duration>2 weeks: 79 points; Previous history of gastrointestinal bleeding: 100 points; The result of CE was positive: 79 points; Specific treatment:-85 points; Minimum hemoglobin before CE<80 g/L: 41 points. The prediction model constructed from the above five variables had good discriminative capability (concordance index=0.798, 95%CI: 0.732-0.865). The calibration curves showed high consistency between nomogram-predicted probabilities and actual observations. The decision curves showed that when the threshold probability was above 0.04, the use of the nomogram to predict rebleeding provided a greater net benefit than the assumption of "all patients rebleeding or no patients rebleeding". Conclusion: The prediction model established in this study has a good ability to predic rebleeding in patients with OGIB after CE examination.

Efficacy of percutaneous cement-augmented screw fixation plus percutaneous kyphoplasty in the management of unstable osteoporotic vertebral compression fractures.

OBJECTIVE: The present study was performed to compare the efficacy of percutaneous kyphoplasty (PKP) vs. percutaneous cement-augmented screw fixation plus PKP in the management of unstable osteoporotic vertebral compression fractures (OVCF). PATIENTS AND METHODS: A total of 197 patients with unstable OVCF treated in the Department of Spine Surgery, Lianyungang First People's Hospital from September 2019 to September 2021 were recruited and assigned via random number table method 1:1 to receive either PKP (group A, n=106) or PKP plus percutaneous cement-augmented screw fixation (group B, n=91). The outcome measures for the evaluation of different surgical methods included visual analogue scale (VAS), the height of the anterior-posterior border of the injured spine, Cobb angle of the posterior convexity, Oswestry disability index (ODI) scores, and Japanese Orthopaedic Association (JOA) scores. RESULTS: PKP exhibited shorter operative time and length of hospital stay and less intraoperative blood loss vs. PKP plus percutaneous cement-augmented screw fixation (p<0.05). Patients with PKP plus percutaneous cement-augmented screw fixation experienced milder postoperative pain vs. those with PKP alone at 7 days postoperatively, as evidenced by the lower VAS scores (p<0.05). PKP plus percutaneous cement-augmented screw fixation provided more restoration of anterior margin height and posterior convexity Cobb angle vs. PKP alone (p<0.05). Patients with PKP only showed slightly higher Japanese Orthopaedic Association (JOA) scores than those with combined surgery, while the postoperative clinical signs between the two arms were similar (p>0.05). CONCLUSIONS: Single PKP features the benefits of minimal trauma, simple operation, and rapid postoperative recovery in the treatment of OVCF. PKP plus percutaneous cement-augmented screw fixation for severe OVCF provided distinctly better performance than PKP alone in terms of early pain relief, restoration of vertebral body height, correction of posterior convexity deformity, and firm spinal stability.

Efficacy of minimally invasive transforaminal lumbar interbody fusion plus cement-augmented pedicle screw fixation in the treatment of degenerative lumbar spine disease with osteoporosis in the elderly.

OBJECTIVE: The aim of this study was to evaluate the efficacy of minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) plus cement-augmented pedicle screw fixation in the treatment of degenerative lumbar spine disease with osteoporosis in the elderly. PATIENTS AND METHODS: From February 2020 to January 2021, 40 elderly patients with degenerative lumbar spine disease with osteoporosis admitted to our hospital were randomly assigned (1:1) to receive either MIS-TLIF plus cement-augmented pedicle screw fixation (group A) or TLIF plus cement augmentation (group B), with 19 cases in group A and 21 cases in group B. Outcome measures included visual analogue scale (VAS), Oswestry Dysfunction Index (ODI) and Japanese Orthopedic Association Scores (JOA), operative duration, intraoperative bleeding, postoperative drainage volume, and the incidence of complications. Frontal and lateral radiographs of the lumbar spine and computed tomography (CT) were performed 3 days after surgery to observe the distribution of bone cement. At 12 months postoperatively, the fusion of the bone graft was evaluated according to the Bridwell intervertebral fusion criteria based on the lumbar frontal and lateral radiographs. RESULTS: All 40 cases completed the surgery successfully and were followed up for 12 months. The two groups did not differ significantly in terms of operative duration (p>0.05). MIS-TLIF plus cement-augmented pedicle screw fixation was associated with significantly less intraoperative bleeding volume (142.25±40.93 mL) and (76.25±17.54 mL) vs. TLIF plus cement augmentation (322.00±93.45 mL, 159.75±54.74 mL) (p<0.05). The difference in the VAS scores, ODI, and JOA scores between the two groups preoperatively and at the final follow-up showed no statistical significance (p>0.05). Patients receiving MIS-TLIF plus cement-augmented pedicle screw fixation had significantly lower VAS scores and ODI and higher JOA scores vs. TLIF plus cement augmentation (p<0.05). The lumbar frontal and lateral radiographs and CT of the two groups 3 days after surgery showed good cement distribution and no cement leakage. At the final follow-up, no complications were seen in group A, and there was one case of intervertebral cement leakage in group B. The intervertebral graft fusion was grade I in both groups. CONCLUSIONS: MIS-TLIF plus cement-augmented pedicle screw fixation shortens the operative time, alleviates postoperative pain, facilitates operative lumbar spine function restoration, and provides favorable intervertebral implant fusion.

Fast ion shuttling which is robust versus oscillatory perturbations.

Shuttling protocols designed by shortcut-to-adiabaticity techniques may suffer from perturbations and imperfect implementations. We study the motional excitation of a single ion shuttled in harmonic traps with time-dependent, 'systematic' oscillatory perturbations around the nominal parameters. These elementary perturbations could form any other by superposition. Robust shuttling strategies are proposed and compared, and optimizations are performed. This article is part of the theme issue 'Shortcuts to adiabaticity: theoretical, experimental and interdisciplinary perspectives'.

Biopsy-tract haemocoagulase injection reduces major complications after CT-guided percutaneous transthoracic lung biopsy.

AIM: To determine whether the injection of haemocoagulase into the biopsy tract can reduce pneumothorax and pulmonary haemorrhage after computed tomography (CT)-guided percutaneous transthoracic lung biopsy (PTLB). MATERIALS AND METHODS: A retrospective study was performed involving patients with undiagnosed pulmonary lesions scheduled for PTLB between January 2020 and March 2021. Patients were assigned to the haemocoagulase group or the non-haemocoagulase group. After CT-guided biopsies were performed with a 17 G coaxial system, patients in the haemocoagulase group received a haemocoagulase injection (0.2-0.5 units) in the biopsy tract as the sheath was withdrawn. Postoperative image studies were performed to evaluate complications, including pneumothorax and pulmonary haemorrhage. Factors, including the patient's position, lesion location, and pathological results, were evaluated to determine their associations with the complications. RESULTS: A total of 100 patients were included, with 44 men and a mean age of 53 years old. The overall incidences of pneumothorax and pulmonary haemorrhage were 15% and 13%, respectively. The incidences of pneumothorax and pulmonary haemorrhage were statistically significantly lower in the haemocoagulase group (8% and 6%, respectively) than in the non-haemocoagulase group (22% and 20%, respectively; p=0.04 and 0.03, respectively). There was no statistically significant difference in haemoptysis between the haemocoagulase (6%) and non-haemocoagulase (2%) groups (p=0.23). There were also no statistically significant associations of pneumothorax or pulmonary haemorrhage with the patients' positions, lesion location, or pathological results. CONCLUSION: Biopsy tract haemocoagulase injection reduced the incidences of postoperative pneumothorax and pulmonary haemorrhage after PTLB.

Combined chemoradiotherapy showed improved outcome with early-stage HPV-positive oropharyngeal cancers.

BACKGROUND: The revised 8th Edition American Joint Committee on Cancer (AJCC) Head and Neck Staging Manual distinguishes HPV-mediated from non-HPV-mediated oropharyngeal cancer (OpSCC). The objective was to analyze OpSCC treatment modalities and outcomes. METHODS: A retrospective study of OpSCC patients treated with radiotherapy or chemoradiotherapy between January 1st, 2000, and December 31st, 2008, as identified from the BC Cancer Registry. All patients received treatment at cancer clinics and had at least 5 years follow-up post-treatment. A total of 1259 OpSCC patients were identified. After initial chart reviews, 288 patients were excluded from further analysis and the majority (n = 198) was due to not receiving curative treatment. Based on the availability of formalin-fixed, paraffin-embedded (FFPE) tissue, patients were divided into two cohorts: Study Cohort (FFPE available, n = 244) and General Cohort (FFPE unavailable, n = 727). The Study Cohort was restaged according to AJCC 8th Edition based on p16 immunohistochemistry status. Kaplan-Meier analysis was used to estimate the 5-year overall survival (OS), disease-specific survival (DSS), and locoregional recurrence-free survival (LFS). RESULTS: Among 971 patients, OpSCC age-adjusted incidence rate was observed to have increased from 2.1 to 3.5 per 100,000 between 2000 and 2008. The General Cohort was relatively older than the Study Cohort (60.1 ± 10.5 vs. 57.3 ± 9.4), but both cohorts were predominantly males (78.3% vs. 76.2%). Amongst the Study Cohort, 77.5% were p16-positive, of whom 98.4% were down staged in the 8th Edition. These early-stage patients showed OS improvement for those treated with chemoradiation, compared to radiation alone (85.8% vs. 73.1%, p = 0.05). CONCLUSIONS: OpSCC incidence is increasing in BC. The addition of chemotherapy to radiotherapy may portend a benefit in OS even for early-stage p16-positive OpSCC. Additional research is necessary to assess the safety of treatment de-escalation even among early-stage disease.

[MiR-4484 regulates the expression of integrin α 6 in gastric cancer tissues and its significance].

Objective: To investigate the relationship between the expression of integrin α 6 (ITGA6), miR-4484 and the pathologic stage of gastric cancer. Methods: Gastric cancer tissues and normal gastric mucosa tissues adjacent to cancer (>5 cm from tumor margin) of 30 patients with primary gastric cancer who underwent direct surgical resection without adjuvant therapy from June to September 2017 in West China Hospital of Sichuan University were selected. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression levels of miR-4484 and ITGA6, western blot was used to detect the expression level of ITGA6 protein, dual luciferase reporter gene was used to verify the relationship between ITGA6 and miR-4484. Spearman's correlation analysis was used to determine the relationship between miR-4484 and ITGA6 expression levels in gastric cancer tissues. Results: The expression level of ITGΑ6 in gastric cancer (32.30±13.47) was higher than that in matched normal gastric tissues (24.55±10.25, P=0.015), the area under the receiver operating characteristic (ROC) curve was 0.660 and the diagnostic sensitivity and specificity were 43.3% and 96.7%, respectively. The expression level of miR-4484 in gastric cancer (4.11±2.87) was lower than that of matched normal gastric tissues (5.75±2.80, P=0.029), the area under the ROC curve was 0.690 and the diagnostic sensitivity and specificity were 30.0% and 86.7%, respectively. The expression level of miR-4484 was negatively correlated with ITGA6 in gastric cancer tissues (r=-0.621, P<0.001). The expression level of ITGA6 protein in gastric cancer tissues (0.65±0.19) was higher than that in normal adjacent tissues (0.26±0.12, P<0.001). Compared with ITGA6 3'UTR wild-type+ miR-NC group, ITGA6 3'UTR wild-type+ miRNA mimics group had lower luciferase activity (50.69±5.10, 34.00±1.19, P<0.001), while the luciferase activity of ITGA6 3'UTR wild-type+ ASO miR-4484 group was higher than that of ITGA6 3'UTR wild-type+ miR-NC group (82.44±6.37, 50.69±5.10, P<0.001), indicated that ITGA6 was the direct target gene of miR-4484. The expression levels of miR-4484 in T1, T2, T3 and T4 (4a and 4b) gastric cancer tissues were 9.98±2.24, 5.28±2.03, 2.92±2.04 and 4.11±2.87, respectively, with statistical significance (P<0.001). The expression levels of ITGA6 in N0, N1, N2 and N3 gastric cancer tissues were 29.55±8.32, 21.71±3.75, 24.60±8.79 and 40.69±15.83, respectively, with statistical significance (P=0.022). The expression levels of miR-4484 in N0, N1, N2 and N3 gastric cancer tissues were 5.01±3.52, 5.48±2.76, 5.88±1.83 and 2.30±1.56, respectively, with statistical significance (P=0.032). The expression levels of ITGA6 in M0 and M1 gastric cancer tissues were 26.28±7.66 and 52.08±8.12, respectively, with statistical significance (P<0.001). The expression levels of miR-4484 in M0 and M1 gastric cancer tissues were 4.95±2.74 and 1.34±0.80, respectively, with statistical significance (P<0.001). Conclusions: ITGA6 is upregulated in gastric cancer tissues, while miR-4484 is downregulated in the gastric cancer group, and its expression level is related to the clinicopathological features of gastric cancer. ITGA6 is the direct target gene of miR-4484, implicates that miR-4484 may inhibit the invasion and metastasis of gastric cancer by regulating the expression of ITGA6. Both miR-4484 and ITGA6 may be the new prognostic markers and potential therapeutic targets of gastric cancer.

[Efficacy and safety of intraoperative ultrasound-assisted neuroendoscopy in the treatment of hypertensive intracerebral hemorrhage via transsylvian approach].

Objective: To explore the efficacy and safety of intraoperative ultrasound-assisted neuroendoscopy for treating hypertensive intracerebral hemorrhage (HICH) via lateral fissure and insula approach. Methods: The clinical data of 66 patients with HICH in basal ganglia who underwent intraoperative ultrasound-assisted neuroendoscopy via lateral fissure and insula approach for hematoma evacuation were retrospectively analyzed, including operative data and follow-up results. All patients had no brain hernia before operation. The hematoma was located by the ultrasound and then the path into the hematoma cavity was determined. Meanwhile, the residual hematoma was also detected by using the ultrasound. The clearance rate of hematoma was observed by CT. Barthel index was used to evaluate the activity of daily living. Results: The amount of residual hematoma was less than 10% in 63 ases and 10%-20% in 3 cases. There were 11 cases of postoperative pneumonia, of whom 9 cases underwent percutaneous tracheotomy. Rebleeding occurred in 2 cases, but the amount was small, and there was no need for reoperation. Meanwhile, there was 1 case of secondary intracranial infection and 1 case of secondary hydrocephalus. No case of cerebral contusion was reported. Half a year after the operation, Barthel Index grading showed that there were 3 cases of grade I, 25 cases of grade Ⅱ, 34 cases of grade Ⅲ, 3 cases of grade Ⅳ and 1 case of death, respectively. Conclusions: Intraoperative ultrasound-assisted neuroendoscopy via lateral fissure and insula approach can improve the efficacy and safety of treatment for HICH.

[Estimating Postmortem Interval by Protein Chip Detection Technology Combined with Multidimensional Statistics].

ABSTRACT: Objective To obtain the protein expression profile of rat liver tissue after death by the 2100 bioanalyzer combined with protein chip, and infer the relationship between protein expression profile and postmortem interval. Methods Rats were killed by abdominal anesthesia and placed at 16 ℃. Water-soluble proteins in liver tissues were extracted at 14 time points after death. The expression profile data of proteins with relative molecular weight of 14 000-230 000 were obtained using protein chip, and principal component analysis （PCA）, partial least squares-discriminant analysis （PLS-DA） and Fisher discriminant were used to analyze the data. Results According to the changes of protein expression profile, the postmortem interval was divided into group A （0 d）, group B （1-9 d）, group C （12-30 d） according to the result of PLS-DA. The prediction accuracy of the training set and test set of the model were all 100.0%, and the internal cross-validation of the training set was 100.0% according to Fisher discriminant. The Fisher discriminant model at each time point of group B and C was established to narrow the time window of postmortem interval estimation. The prediction accuracy of the training set and test set were all 100.0%, and the internal cross-validation accuracy of the training set was 100.0% in group B. The prediction accuracy of the training set and test set were respectively 95.2% and 78.6% in group C, and the internal cross-validation of the training set was 88.1%. Conclusion Protein chip detection technology can quickly and easily obtain the expression profile of water-soluble proteins of rat liver tissue with a relative molecular weight of 14 000-230 000 at different time points after death. PLS-DA and Fisher discriminant models are established to classify and predict the postmortem interval, in order to provide new ideas and methods for postmortem interval estimation.

Determination of Escitalopram in Biological Samples by Dispersive Liquid-Liquid Microextraction Combined with GC-MS/MS.

ABSTRACT: Objective To establish a method for determination of escitalopram in biological samples by ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry （GC-MS/MS） and provide evidences for forensic determination of cases related to escitalopram. Methods The 1-hexyl-3-methylimidazolium hexafluorophosphate （[C6MIM][PF6]） was selected as an extract solvent to process biological samples. Ultrasound-assisted extraction was used on the samples. Then the samples were detected by GC-MS/MS. Results The linear range of escitalopram in blood and liver were 5.56-1 111.10 ng/mL and 0.025-5.00 mg/g, respectively. The correlation coefficient （r） were greater than 0.999, limit of detection （LOD） were 4.00 ng/mL and 2.00 µg/g, limit of quantitation （LOQ） were 14.00 ng/mL and 6.00 µg/g, respectively. The extraction recovery rates were all greater than 50%, the interday and intraday precision were less than 20%. Escitalopram was detected in blood and liver samples from the actual poisoning case by this method with a content of 1.26 µg/mL and 0.44 mg/g, respectively. Conclusion The ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with GC-MS/MS is environment friendly, rapid, has good enriching effect and consumes less organic solvent and can be used for forensic determination of escitalopram related cases.

[Preliminary application of postoperative fast track transfer to intensive care unit for the geriatric hip fractures under enhanced recovery after surgery].

Objective: To develop a fast track transfer to intensive care unit (ICU) for the perioperative high-risk elderly patients after hip fracture surgery and analyze the preliminary clinical effect of the application. Methods: From January 2014 to December 2017, before the application of postoperative fast track transfer to ICU, the clinical data of 195 elderly patients with hip fracture were included in a retrospective analysis. Among 195 hip fracture patients, 18 were transferred to ICU post operation (non-fast track group). Multivariate logistic regression analysis was applied to investigate relevant risk factors for transferring to ICU after hip fracture surgery. Based on risk factors acquired from the analysis and clinical experience, the fast track transfer to ICU for the perioperative high-risk elderly patients after hip fracture surgery was constructed according to the preliminary and experiential criteria. From January 2018 to December 2019, the clinical data of 70 patients (fast track group) who were transferred to ICU after hip fracture surgery through the fast track were collected and compared with non-fast track group. Results: Multivariate regression analysis revealed that American Society of Anesthesiologists classification(≥Ⅲ) (OR=4.260, 95%CI:1.157-15.683, P=0.029), pre-hospital stage (≥48 h) (OR=4.301, 95%CI:1.212-15.266, P=0.024), hemoglobin concentration at admission(<90 g/L) (OR=7.979, 95%CI:1.936-32.889, P=0.004), coronary heart disease as one comorbidity(OR=6.063, 95%CI:1.695-21.693, P=0.006) were independent risk factors for transferring to ICU after hip fracture surgery. There were no significant difference in gender, age, fracture type, hemoglobin concentration at admission and time of pre-hospital stage between the non-fast track group and fast track group(all P>0.05). However, the number of comorbidities in the fast track group was significantly higher than that in the non-fast track group (Z=-1.995, P=0.046). The time to surgery, postoperative hospital stay, and length of hospital stay in fast track group were all significantly less than those in non-fast track group (Z=-2.121, -2.726, -3.130, all P<0.05). Also, there were fewer medical consultations needed and fewer patients who stayed in ICU more than or equal to 2 nights in fast track group than that in non-fast track group(all P<0.05). There were no significant difference in the rate of patients who transferred from the general ward to ICU after transferring from ICU to the general ward, the proportion of patients who received more than or equal to 4 departments, operation time, hospitalization expense, mortality during hospitalization, 30-day mortality and 90-day mortality after operation between the two groups(all P>0.05). Conclusions: The fast track constructed in this study can reduce time to surgery, postoperative hospitalization stay and length of hospitalization stay for the perioperative high-risk elderly patients with hip fractures and is a specific clinical application of eras concept based on multidisciplinary team.

[Diagnostic value of digital breast tomosynthesis for mass lesions in dense breast].

Objective: To explore the diagnostic efficiency of digital breast tomosynthesis (DBT) for mass lesions in dense breast by comparing with digital mammography (DM). Methods: A retrospective analysis was made of 125 female patients with breast masses confirmed by pathology in the First Affiliated Hospital of Jinzhou Medical University from June 2018 to April 2019, all of whom were dense breast. Pathological results were taken to compare the diagnostic value of DM, DBT and the combination of the two for breast mass lesions. Results: The overall detection rates of DM, DBT and their combination were 77.6% (97/125), 88.8%(111/125) and 92.8%(116/125) in dense breast. The detection rates of burr sign and lobulation sign by DM and DBT in malignant tumors were 39.7%(31/78), 60.3%(47/78) and 48.7%(38/78), 67.9%(53/78), respectively, with statistically significant differences (all P<0.05). The sensitivity of DM, DBT and their combination in differentiating benign from malignant breast mass lesions of dense breast was 75.6% (59/78), 83.3%(65/78), 93.6%(73/78), the specificity was 63.8%(30/47), 80.9% (38/47), 76.6%(36/47),and the area under ROC curve(AUC) was 0.697, 0.821 and 0.852, respectively. Conclusion: DBT is more effective than DM in the diagnosis of dense breast mass lesions, and the combination of DBT and DM is more significant.

Coagulation factor XIIIa cross-links amyloid ß into dimers and oligomers and to blood proteins.

In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid ß (Aß) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aß in CAA, the ability of FXIIIa to cross-link Aß has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aß40 into dimers and oligomers (kcat/Km = 1.5 × 105 m-1s-1), as well as to fibrin, platelet proteins, and blood clots under flow in vitro Aß40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that FXIIIa-mediated cross-linking may contribute to the formation of Aß deposits in CAA and Alzheimer's disease.

Using ddPCR to assess the DNA yield of FFPE samples.

OBJECTIVES: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples. METHODS: We used 10 µm-thick sections from 5 FFPE oral tumoral blocks, each consisting of 10-15 sections. The protocol variables tested included: 1) tissue staining; 2) duration and 3) temperature of post-digestion heat treatment; and 4) DNA extraction method. DNA quantity was assessed using the NanoDrop 2000 (Thermo Fisher Scientific, USA), the Qubit fluorometer (Thermo Fisher Scientific, USA), and a ddPCR-based assay. DNA quality was assessed using a ddPCR assay for the degree of fragmentation and the effectiveness of removing crosslinks with varying guanine-cytosine (GC)-content. RESULTS: Deparaffinization with xylene helped to increase the DNA yield. Tissue staining (methyl green staining, pH 6) prior to microdissection, comparing to no staining, caused additional DNA fragmentation. Compared to column-based method, DNA extracted with phenol chloroform and ethanol precipitation increased the degree of fragmentation and lowered the yield of amplifiable DNA. The cross-linking derived from GC-contents may not be the only factor impacting on the DNA quality. CONCLUSIONS: Samples undergoing different pre-treatment conditions prior to extraction can impact the yield of amplifiable DNA. Our ddPCR assay can be used to assess for both DNA quantity and quality.

Potential clinical implications of HPV status and expressions of p53 and cyclin D1 among oropharyngeal cancer patients.

BACKGROUND: There is increasing evidence that high-risk human papillomavirus plays significant role in oropharyngeal cancer; however, there is lack of knowledge on the interplay between the virus and its downstream-related molecules and their possible prognostic values. The objectives of the study are to better understand the interplay of the HR-HPV and its associated downstream molecules and to evaluate potential biomarkers for patient outcomes. METHODS: We conducted a retrospective study with available formalin-fixed, paraffin-embedded tissue from 244 oropharyngeal cancer patients that received curative radiotherapy or concurrent chemoradiotherapy from 2000 to 2008. In addition to chart review, we performed HPV DNA and RNA in situ hybridization and immunohistochemistry for p53, the retinoblastoma protein, p16, and cyclin D1 analysis. Cox proportional hazard and Kaplan-Meier survival analysis were used to determine the prognostic markers for clinical outcomes. RESULTS: Patients averaged 57.3 ± 9.4 year-old and were mostly males (76.2%) and ever-smokers (76.2%). All patients received curative radiotherapy, and 44.3% received concurrent chemoradiotherapy. We detected the human papillomavirus in 77.9% of study patients. Ever-smokers, more advanced tumor stage, and receiving radiotherapy only had poorer 5-year overall survival, disease-specific survival, and loco-regional recurrence. Cases with positive human papillomavirus and p53 overexpression had poorer disease-specific survival. Cases without human papillomavirus, but cyclin D1 overexpression, were associated with poorer 5-year overall survival. CONCLUSIONS: Our data suggest that additional p53 and cyclin D1 testing may benefit oropharyngeal cancer patients with known human papillomavirus status.

Altered Immune-Related Gene Expressions Indicate Oral Cancer Nodal Disease.

Lymph nodal disease (LN+) is the most significant prognostic factor of oral squamous cell carcinoma (OSCC). Current risk indicator(s) for guiding elective neck dissection (END) is insufficient for clinically node-negative (cN0) patients, resulting in under- or overtreatment. While the role of immunological events in tumorigenesis and metastasis is evident, the prognostic implication in OSCC remains unclear. The study objective was to investigate large-scale immune-related gene expression and determine its prognostic value on node-free survival (NFS). We analyzed patients who received intent-to-cure surgery with at least 3 y of follow-up and known outcome of LN through a pan-Canadian surgical trial. Total RNA was extracted from surgical tissues with >70% tumor content and analyzed on a 730-gene panel (NanoString nCounter® PanCancer Immune Panel). We first profiled gene expression in a fresh-frozen (FF) discovery set to identify differentially expressed (DE) genes, which were then used in unsupervised clustering analysis to identify patient subgroups. The prognostic value of the identified DE genes was then validated on formalin-fixed, paraffin-embedded (FFPE) samples. A total of 177 RNA samples were derived from 89 FF and 88 FFPE surgical tissues, of which 45 (51%) and 40 (45%), respectively, were from patients who developed LN+. We identified 6 DE genes overexpressed in LN+ tumors (false discovery rate <0.001; log2 fold change >1). Clustering analysis separated the patients into 2 subgroups (CM1, CM2), with CM2 exhibiting significantly increased expression and worse 5-y NFS rate (28%; P < 0.001). The prognostic value of these 6 candidate genes was validated on FFPE samples, which were also separated into 2 distinct prognostic groups, confirming the association between increased gene expression and poor 5-y NFS (CM1, 70.3%; CM2, 43.3%; P = 0.01). This is the first study identifying a panel of immune-related genes associated with NFS that can potentially be used clinically stratifying the risk of LN+ at the time of OSCC diagnosis.

[Comparison of the Skin and Skeletal Muscle Contusion in Rats Induced by Blunt Force with Different Heights].

OBJECTIVES: To explore the differences in the repair process of skin and skeletal muscle after contusion caused by blunt force attack with different heights. METHODS: Three degrees of contusion were performed on SD rats' right hind limbs by a designed free-dropping device falling from 15, 30 and 50 cm heights, which as a main consideration factor for degree of injury. The repair process of skin and skeletal muscle at 6 h, 24 h, 3 d, 7 d and 13 d after contusion were observed using routine histological methods. RESULTS: Hematoma within skin and/or muscle was found in the rats' hind limbs after contusion with three different heights. The repair processes were similar at 24 h after contusion. However, with the increase of height, the display degree was more obvious. At 3 d after contusion, the RBC of the hemorrhagic region would be decomposed and elapsed in 15 cm contusion group, but for 30 cm contusion group, it delayed to 7 d. At 13 d after contusion, the similar result was found in 15 cm and 30 cm contusion groups, in contrast, the 50 cm contusion group was still in the proliferative phase. CONCLUSIONS: With the increase of height, the occurring rate of hematoma within skin and muscle at the same time increases, and the more serious histological appearance after contusion, including inflammation and proliferation, the longer healing process are observed. According to the results of present study and considering forensic application, the contusion model with 50 cm height （2.58 J/cm²ï¼‰ is recommended as the experimental animal model for the future study of wound age estimation on contusion.

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

An actionable test using loss of heterozygosity in identifying high-risk oral premalignant lesions.

OBJECTIVES: To develop an actionable test using fluorescence capillary electrophoresis (FCE) to assess loss of heterozygosity (LOH) of histologically similar low-grade lesions (LGLs) to identify high-risk lesions for oral cancer progression. STUDY DESIGN: To determine the cutoffs of LOH, the FCE results of 52 surgical margin samples were used to compare with the existing LOH results from the previously validated 32 P-GE approach. Using the developed FCE workflow, an independent set of 102 LGLs with known progression status was used to determine the LOH molecular risk (MR) patterns and associated risk of progression. RESULTS: Using 65% cutoff LOH-FCE, the agreement of LOH-32 P-GE had an average of 82.3% (76.8-87.8). Compared with nonprogressors (n = 61), anatomic site and MR patterns (LOH at 9 p21, 3 p14, or 17 p13) were independent risk factors. High-risk profile of tongue and MR3 (LOH at 9 p21 and/or 3 p14 and 17 p13) was significantly associated with progression (hazard ratio [HR] 6.7; 95% confidence interval [CI] 2.6-17.6) with specificity of 98.4% at identifying progressors. CONCLUSIONS: We have developed an objective test using LOH to stratify the risk of LGLs. With further validation, it can be used in the clinical settings to provide clinicians additional information guiding the management of these lesions.