Genomic Signature of Mismatch Repair Deficiency in Areca Nut-Related Oral Cancer.

Areca nut (AN) chewing contributes to an increase of oral squamous cell carcinoma (OSCC) cases in South and Southeast Asia; however, genomic events underlying the carcinogenesis process of AN-related OSCC remain unclear. Here, we comprehensively describe the genomic and transcriptome alterations of 113 Chinese OSCC patients (89 AN related and 24 AN negative) by whole-exome sequencing and RNA sequencing, and we compared the genomic differences between AN-related and AN-negative samples by integrating sequencing data of 325 OSCC patients from The Cancer Genome Atlas database and 50 from a published Taiwanese study. We identified 11 significantly mutated genes for OSCC, including 4 novel ones (ATG2A, WEE1, DST, and TSC2), of which WEE1 and ATG2A mutated with significantly higher rates in AN-related samples (P = 0.04 and P = 0.003, respectively). Mutational signature analysis revealed that AN-related OSCCs were specially characterized by the genomic signature of mismatch repair deficiency (dMMR), which could also predict the prognosis status of AN-related OSCC. In addition, an elevated PD-L1 expression was also observed in both AN-related patients (P = 3.71 × 10-11) and those with a high dMMR level (P = 1.99 × 10-4). Further differential expression analysis and in vitro experiments confirmed the role of dMMR in the development of OSCC induced by AN exposure. Taken together, this study first revealed the molecular profiles and highlighted the role of dMMR in AN-related OSCC among the Chinese population and identified that AN-related OSCC may represent a potential cohort for effective anti-PD-1/L1 immunotherapy.

Incorporation of log odds of positive lymph nodes into the AJCC TNM classification improves prediction of survival in oral cancer.

OBJECTIVES: To assess the prognostic performance of a new N classification that incorporates the log odds of positive lymph nodes (LODDS) into the routinely used pathological N classification for oral squamous cell carcinoma (OSCC) patients. DESIGN: Retrospective cohort study utilising LODDS into pN category was performed, and the AJCC TNM stage and T-New N-M stage were compared with respect to 5-year disease-specific survival (DSS) rates. The discriminability was evaluated from the linear trend chi-square test, Akaike information criterion (AIC) and Harrell's c-statistic. SETTING: Medical centrer in Taiwan. PARTICIPANTS: A total of 463 patients received primary surgery and neck dissection between 2004 and 2013 for OSCC. MAIN OUTCOME MEASURES: The discriminability for 5-year DSS rates. RESULTS: The median follow-up period was 54 months, the mean patient age was 54 ± 11 years and 428 patients (92.4%) were male. The patients with higher LODDS had worse 5-year DSS rates. Incorporation of LODDS into the prognostic model based on the seventh edition of the TNM classification significantly improved discriminative performance for 5-year DSS with a lower AIC (1883 versus 1897), and higher prediction accuracy (Harrell's c-statistic: 0.768 versus 0.764). CONCLUSIONS: By utilising a merger of the LODDS and pN classifications to create a new N classification has better discriminatory and predictive ability than pathological TNM staging and could help identify high-risk patients for intense adjuvant therapy.

Correlation between liver cirrhosis and risk of death from oral cancer: Taiwan cohort study.

BACKGROUND: A nationwide population-based cohort was used to examine the severity of liver cirrhosis and risk of mortality from oral cancer. METHODS: The cohort consisted of 3583 patients with oral cancer treated by surgery between 2008 and 2011 in Taiwan. They were grouped on the basis of normal liver function (n = 3471), cirrhosis without decompensation (n = 72) and cirrhosis with decompensation (n = 40). The primary endpoint was mortality. Hazard ratios of death were also determined. RESULTS: The mortality rates in the respective groups were 14.8 per cent, 20.8 per cent and 37.5 per cent at one year (p < 0.001). The adjusted hazard ratios of death at one year for each group compared to the normal group were 2.01 (p = 0.021) for cirrhotic patients without decompensation, 4.84 (p < 0.001) for those with decompensation and 2.65 (p < 0.001) for those receiving chemotherapy. CONCLUSION: Liver cirrhosis can be used to predict one-year mortality in oral cancer patients. Chemotherapy should be used with caution and underlying co-morbidities should be managed in cirrhotic patients to reduce mortality risk.

Experimental study of fat grafting under negative pressure for wounds with exposed bone.

BACKGROUND: The combination of fat grafting and negative pressure (VAC) therapy represents a synergistic interaction of all essential components for wound healing. The aim of this experimental study was to determine whether it could promote healing of wounds with exposed bone. METHODS: Full-thickness wounds with denuded bone in Sprague-Dawley rats were treated with either polyurethane foam dressing, fat grafting alone, polyurethane foam dressing with VAC, or polyurethane foam dressing with VAC combined with a single, or two administrations of fat graft. Wound healing kinetics, tissue growth, cell proliferation (Ki-67) and angiogenesis (platelet endothelial cell adhesion molecule 1 and α-smooth muscle actin) were investigated. Messenger RNA levels related to angiogenesis (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF)), profibrosis (platelet-derived growth factor A and transforming growth factor ß), adipocyte expression (fatty acid-binding protein (FABP) 4 and peroxisome proliferator activated receptor γ), and extracellular matrix remodelling (collagen I) were measured in wound tissues. RESULTS: Wounds treated by VAC combined with fat grafting were characterized by cell proliferation, neoangiogenesis and maturation of functional blood vessels; they showed accelerated granulation tissue growth over the denuded bone compared with VAC- or foam dressing-treated wounds. Fat grafting alone over denuded bone resulted in complete necrosis. Expression of angiogenesis markers (VEGF and b-FGF) and adipocyte expression factors (FABP-4) was upregulated in wounds treated with VAC combined with fat grafting. CONCLUSION: Fat grafting with VAC therapy may represent a simple but effective clinical solution for a number of complex tissue defects, and warrants testing in clinical models. SURGICAL RELEVANCE: The combination of fat grafting and vacuum therapy represents a synergistic interaction of regenerative cells, hospitable wound matrix and stimulating micromechanical forces. It could accelerate complex wound healing through cell proliferation, neoangiogenesis and maturation of functional blood vessels. The efficacy of a multimodal wound healing approach is established in this experimental model; it could easily be translated into clinical trials of treatment for difficult wounds.

MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma.

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules of about 20-23 nucleotides in length, which negatively regulate protein-coding genes at post-transcriptional level. Using a stem-loop real-time-PCR method, we quantified the expression levels of 270 human miRNAs in 13 nasopharyngeal carcinoma (NPC) samples and 9 adjacent normal tissues, and identified 35 miRNAs whose expression levels were significantly altered in NPC samples. Several known oncogenic miRNAs, including miR-17-92 cluster and miR-155, are among the miRNAs upregulated in NPC. Tumour suppressive miRNAs, including miR-34 family, miR-143, and miR-145, are significantly downregulated in NPC. To explore the roles of these dysregulated miRNAs in the pathogenesis of NPC, a computational analysis was performed to predict the pathways collectively targeted by the 22 significantly downregulated miRNAs. Several biological pathways that are well characterised in cancer are significantly targeted by the downregulated miRNAs. These pathways include TGF-Wnt pathways, G1-S cell cycle progression, VEGF signalling pathway, apoptosis and survival pathways, and IP3 signalling pathways. Expression levels of several predicted target genes in G1-S progression and VEGF signalling pathways were elevated in NPC tissues and showed inverse correlation with the down-modulated miRNAs. These results indicate that these downregulated miRNAs coordinately regulate several oncogenic pathways in NPC.

MEG localization of rolandic spikes with respect to SI and SII cortices in benign rolandic epilepsy.

The purpose of this study was to study the relationship between interictal spike sources and somatosensory cortices in benign rolandic epilepsy of childhood (BREC) using a whole-scalp neuromagnetometer. We recorded spontaneous magnetoencephalography (MEG) and EEG signals and cortical somatosensory-evoked magnetic fields (SEFs) to electric stimulation of the median nerve in 9 children with BREC. Interictal rolandic discharges (RDs) and SEFs were analyzed by equivalent current dipole (ECD) modeling. Based on the orientation and locations of corresponding ECDs, we compared generators of RDs with primary (SI) and second somatosensory cortices (SII). Our results showed that RDs and SII responses had similar ECD orientation on the magnetic field maps. The ECDs of RDs were localized 15.3 +/- 1.9 and 12.2 +/- 2.8 mm anterior to SI and SII, respectively. The spatial distance on average from the location of RDs to SII (21.9 +/- 1.6 mm) cortex was significantly shorter than to SI cortex (29.7 +/- 1.7 mm) (P<0.01, Wilcoxon signed-rank test). In conclusion, the cortical generators for RDs in patients with BREC are localized in the precentral motor cortex, closer to hand SII than to SI cortex.

Differentiation of recurrent nasopharyngeal carcinoma and skull base osteoradionecrosis by Epstein-Barr virus-derived latent membrane protein-1 gene.

OBJECTIVES: In patients with nasopharyngeal carcinoma (NPC), the differentiation between recurrent primary cancer and osteoradionecrosis (ORN) is clinically difficult. Epstein-Barr virus (EBV)-derived latent membrane protein-1 (LMP-1) has been demonstrated to be highly associated with NPC. The objective of this study is to define the role of the LMP-1 gene in the differential diagnosis of recurrent NPC and ORN. STUDY DESIGN: Prospective. METHODS: From July 1998 to June 2000, 15 postirradiated patients with NPC who were initially diagnosed to have skull base ORN underwent endoscopic sequestrectomy. The sequestra were examined for the presence of the LMP-1 gene and cancer. RESULTS: Two of 15 patients had recurrent cancer and only these two patients demonstrated a positive LMP-1 gene in their surgically removed sequestra. The presence of the LMP-1 gene in the sequestrum coincided with biopsy-proven local recurrence. CONCLUSIONS: The LMP-1 gene is a potential marker to differentiate between recurrent NPC and ORN. The presence of the LMP-1 gene in patients with ORN may indicate local recurrence.

Endoscopic excision of benign tumors in the forehead and brow.

Twenty-five cases of benign tumor of the forehead and brow were excised successfully with endoscope-assisted surgery. The access incision was selected strategically behind the front hairline. For tumors in the middle of the forehead, the tumor was approached by subgaleal dissection. For those in the brow or temporal area, the dissection plane was just superficial to the deep temporal fascia. Patient age ranged from 3 to 59 years. The mass varied in size from 1.0 x 0.5 to 2.0 x 2.0 cm. There were 18 lipomas, 6 dermoid cysts, and 1 pilomatricoma. There was no residual mass or recurrence 1 to 24 months postoperatively. There was no paresthesia or numbness in the scalp. Patients and their families were greatly satisfied with this operation and the absence of visible scarring.

Measurement of the volume of the pedicled TRAM flap in immediate breast reconstruction.

The transverse rectus abdominis musculocutaneous (TRAM) flap is now accepted as the standard for breast reconstruction, but achieving symmetrical breast reconstruction is still a challenge. A precise estimate of the volume of the flap is necessary to reconstruct a symmetrical and aesthetically pleasing breast. Many methods have been developed to overcome this problem, but they have not been suitable for the pedicled TRAM flap. By using a self-made device based on the Archimedes' principle, the authors can calculate accurately the volume of the pedicled TRAM flap and predict reliably the breast volume intraoperatively. The "procedure" is based on a self-made box into which the pedicled TRAM flap is placed. Warm saline is added to the box and the flap is then removed. Flap volume is calculated easily by determining the difference between the preestimated volume of the box and the volume of the residual water. From February to May 2000, this method was used on 28 patients to predict breast volume for breast reconstruction. This study revealed that the difference of the maximal chest circumferences (the index of the breast volume) demonstrates a positive correlation with the difference of the volumes and weights between the mastectomy specimen and the net TRAM flap. However, a more closely positive correlation exists between the differences of maximal chest circumference volume (r = 0.677) than maximal chest circumference weight (r = 0.618). These data reveal that the reconstructed breast's volume has a closer relationship with the volume of the net pedicled TRAM flap, rather than with its weight.

Endoscope-assisted parotidectomy for benign parotid tumors.

Sixteen cases of parotidectomy of benign tumors were performed by means of a short incision with the assistance of endoscopic surgery. The average length of the preauricular and retromandibular incision was 6.9 cm (range, 6.0-8.1 cm). This incision provided safe dissection of the facial nerve trunk and exposed a major part of the gland to direct vision. Identification and separation of the facial nerve branches in peripheral glandular tissue was performed by means of endoscopic surgery instead of wide exposure with a long incision. Because endoscopic surgery provided good illumination and magnification on the monitor, the operation could proceed clearly and sharply in a bloodless field. Therefore, the incidence of facial nerve injury and perioperative morbidity was minimized. In this series, tumors were removed completely, without injury to the facial nerve. Patients were also satisfied with the short and inconspicuous postoperative scars.

Postcallosotomy seizure outcome in hemiconvulsion-hemiatrophy-epilepsy syndrome.

Recently, three patients with hemiconvulsion-hemiplegia-epilepsy syndrome who underwent callosotomy were monitored for more than four years. All patients had atrophy of the right brain hemisphere with left hemiparesis and seizures. Two cases were probably the result of traumatic intracranial hemorrhage and one was due to an unknown cause. Wada tests were done in cases 1 and 2, which showed spared motor function of the atrophic hemisphere, supporting the choice of callosotomy instead of hemispherectomy. Patient 1 had atypical absence seizures and asymmetric generalized tonic seizures before surgery, the latter of which decreased by about 60% after callosotomy. Patient 2 had simple partial seizures of the motor type and complex partial seizures, the latter of which were also induced by touch (somatosensory-induced reflex epilepsy). This patient's complex partial seizures disappeared completely, but the simple partial seizures remained unchanged. Patient 3 had generalized tonic seizures, simple partial seizures of the sensory type and complex partial seizures. After surgery, the frequency of the generalized tonic seizures decreased more than 90%. The simple partial seizures of the sensory type remained unchanged. There were two new types of seizures after surgery, simple partial seizures of the motor type and brief generalized myoclonic jerks. All patients had significant reductions in numbers of seizures of more than 50%.

Endoscopic management of skull base osteoradionecrosis.

OBJECTIVE: Osteoradionecrosis is one of the most serious complications in radiotherapy of nasopharyngeal carcinoma. We describe a new endoscopic approach to resolve resultant skull base osteoradionecrosis. The objective of this study is to evaluate the efficacy of endoscopic management of skull base osteoradionecrosis. STUDY DESIGN: A prospective study of the outcome of endoscopic management for patients with skull base osteoradionecrosis. METHODS: Between 1994 and 1998 six patients who had irradiation previously for nasopharyngeal carcinoma had skull base osteoradionecrosis. A sinoscopic approach was applied for diagnosis and sequestrectomy. This diagnosis was based on the criterion of exposed necrotic bone after removing all crust in the nasopharynx and further confirmed on pathological examination after sequestrectomy. Effective cure was defined as intact mucosal coverage without any ulcer or exposed necrotic bone observed in the nasopharynx and the absence of antecedent accompanying symptoms after management. RESULTS: Six patients (10%) were symptom free. Five (83.3%) patients had effective cure. There was no surgical morbidity or mortality. CONCLUSION: Endoscopic sequestrectomy is a justified approach to skull base osteoradionecrosis.

Postoperative seizure outcome after corpus callosotomy in reflex epilepsy.

Flickering light and color patterns, reading, language, movement, decision making, eating, tapping and touching, hot water immersion and auditory stimulation can induce seizures in some epileptic patients. These are known as the "reflex epilepsies". The mechanism of reflex epilepsy is not clear. Recently, we performed anterior two-thirds corpus callosotomies in two reflex epilepsy patients (ages 12 and 14 years), with follow-up for more than three years. Patient 1 had Lennox-Gastaut syndrome with auditory-induced generalized atonic or tonic seizures (startle epilepsy), which decreased by 60% after callosotomy. Patient 2 had Lennox-Gastaut syndrome with somatosensory-induced generalized tonic seizures (tap epilepsy). He was seizure-free for one year immediately after callosotomy, but his seizures recurred with the same degree and frequency as before surgery. The nonsignificant postoperative seizure outcome suggests that the corpus callosum only plays a partial role in seizure generation. Our report also discusses the possible mechanisms of generation of reflex seizures.

Endoscope-assisted correction of primary varicose veins.

The purpose of this study is to evaluate the possibilities and results of application of endoscopic surgery in the operation of primary varicose veins. With good illumination and magnified viewing, the varicosities, incompetent perforating veins, and healthy veins were clearly visualized. The incompetent perforating veins were clipped and divided securely. The varicose veins and tributaries were dissected and removed completely after being clipped and divided. The healthy veins were preserved if possible. Primary varicose veins in 43 limbs of 37 patients were operated. The operation was conducted through one access incision in 31 limbs (72%), two incisions in 11 limbs (25%), and three incisions in 1 limb. The mean number of incisions was 1.3 in each limb. Hematoma formation occurred in 1 patient due to the slipping of one clip on the third postoperative day. The other patients had uneventful postoperative courses. All 37 patients were reviewed 4 to 30 months postoperatively. Only one limb had recurrent varices at a new site. Ninety-seven percent of limbs (42 of 43) had no recurrence of varicose veins. Seventy-eight percent of patients (29 of 37) were extremely pleased with this operation. Although the other 22% of patients (8 of 37) appreciated this operation, they were not completely satisfied because some preoperative complaints persisted. In this series, the recurrent rate of varicose veins was low (1 in 43 limbs) and postoperative scarring was minimized, in addition to the advantages of endoscopic surgery. These results demonstrate that endoscopic surgery is a worthy alternative procedure for correcting primary varicose veins.

Seizure outcome after corpus callosotomy: the Taiwan experience.

From September 1989 to August 1996, we performed anterior corpus callosotomy in 83 patients. Unfortunately, 9 patients were lost to follow-up. Among the remaining 74 patients, 59 had Lennox-Gastaut syndrome (evolved from infantile spasms in 22), 9 had complex partial seizures with or without secondary generalized seizures, 1 had multifocal independent epileptogenic foci (MISF) syndrome, 3 had hemiconvulsion-hemiplegia-epilepsy (HHE), and 2 had infantile spasms. All cases were followed up for at least 2 years after surgery. The highest rate of significant improvement (more than 50% reduction in seizure frequency) was noted in the patients with generalized tonic-clonic seizures, 82.1% of whom experienced significant improvement, followed by those with generalized tonic seizures (76. 7%), atonic seizures (72.7%), myoclonic seizures (64.9%), atypical absences (58.6%), and complex partial seizure with or without secondary generalization (61.5%). Complete freedom from seizures was noted in 14 cases (18.9%). One patient had the anterior half of his right palm amputated following radial artery thrombosis complicated by insertion of an arterial line during anesthesia. Otherwise, there were no major postoperative complications except for brief mutism and multifocal jerks in some patients during the 1st postoperative week. Thus, we conclude that corpus callosotomy is a safe alternative treatment for all kinds of medically intractable seizures, especially generalized epilepsy.

Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 level in promastigotes and their infection of macrophages in vitro.

The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into a Leishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.

Interaction of Leishmania gp63 with cellular receptors for fibronectin.

The most abundant protein on the surface of the promastigote form of the protozoan parasites Leishmania spp. is a 63-kDa molecule, designated gp63 or leishmanolysin. Because gp63 has been shown to possess fibronectin-like properties, we examined the interaction of gp63 with the cellular receptors for fibronectin. We measured the direct binding of Leishmania to human macrophages or to transfected mammalian cells expressing human fibronectin receptors. Leishmania expressing gp63 exhibited modest but reproducible adhesion to human macrophages and to transfected CHO cells expressing alpha4/beta1 fibronectin receptors. In both cases, this interaction depended on gp63 but occurred independently of the SRYD sequence of gp63, because parasites expressing gp63 with a mutated SRYD sequence bound to macrophages and alpha4/beta1 receptor-expressing cells as well as did wild-type parasites. The contribution of gp63 to parasite adhesion was more pronounced when the assays were performed in the presence of complement, suggesting that the receptors for complement and fibronectin may cooperate to mediate the efficient adhesion of parasites to macrophages. The interaction of gp63 with fibronectin receptors may also play an important role in parasite internalization by macrophages. Erythrocytes to which gp63 was cross-linked were efficiently phagocytized by macrophages, whereas control erythrocytes opsonized with complement alone bound to macrophages but remained peripherally attached to the outside of the cell. Similarly, parasites expressing wild-type gp63 were rapidly and efficiently phagocytized by resting macrophages, whereas parasites lacking gp63 were internalized more slowly. This rapid internalization of gp63-expressing parasites was dependent on the beta1 integrins, because pretreatment of macrophages with monoclonal antibodies to the beta1 integrins decreased the internalization of gp63-expressing parasites. These observations indicate that complement receptors are the primary mediators of parasite adhesion; however, maximal parasite adhesion and internalization may require the participation of the beta1 integrins, which recognize fibronectin-like molecules such as gp63 on the surface of the parasite.

Roles of free GPIs in amastigotes of Leishmania.

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.