Epigenetic Deregulation of Protocadherin PCDHGC3 in Pheochromocytomas/Paragangliomas Associated With SDHB Mutations.

CONTEXT: SDHB mutations are found in an increasing number of neoplasms, most notably in paragangliomas and pheochromocytomas (PPGLs). SDHB-PPGLs are slow-growing tumors, but ∼50% of them may develop metastasis. The molecular basis of metastasis in these tumors is a long-standing and unresolved problem. Thus, a better understanding of the biology of metastasis is needed. OBJECTIVE: This study aimed to identify gene methylation changes relevant for metastatic SDHB-PPGLs. DESIGN: We performed genome-wide profiling of DNA methylation in diverse clinical and genetic PPGL subtypes, and validated protocadherin γ-C3 (PCDHGC3) gene promoter methylation in metastatic SDHB-PPGLs. RESULTS: We define an epigenetic landscape specific for metastatic SDHB-PPGLs. DNA methylation levels were found significantly higher in metastatic SDHB-PPGLs than in SDHB-PPGLs without metastases. One such change included long-range de novo methylation of the PCDHA, PCDHB, and PCDHG gene clusters. High levels of PCDHGC3 promoter methylation were validated in primary metastatic SDHB-PPGLs, it was found amplified in the corresponding metastases, and it was significantly correlated with PCDHGC3 reduced expression. Interestingly, this epigenetic alteration could be detected in primary tumors that developed metastasis several years later. We also show that PCDHGC3 down regulation engages metastasis-initiating capabilities by promoting cell proliferation, migration, and invasion. CONCLUSIONS: Our data provide a map of the DNA methylome episignature specific to an SDHB-mutated cancer and establish PCDHGC3 as a putative suppressor gene and a potential biomarker to identify patients with SDHB-mutated cancer at high risk of metastasis who might benefit from future targeted therapies.

Clinical significance and peculiarities of succinate dehydrogenase B and hypoxia inducible factor 1α expression in parasympathetic versus sympathetic paragangliomas.

BACKGROUND: Succinate dehydrogenase subunit B (SDHB) immunohistochemistry was considered a valuable tool to identify patients with inherited paraganglioma/pheochromocytoma (PGL/PCC). However, previous studies jointly analyzed 2 related but clinically distinct entities, parasympathetic head and neck paragangliomas (HNPGLs) and sympathetic PCCs/PGLs. Additionally, a role for hypoxia inducible factor-1α (HIF-1α) as a biomarker for succinate dehydrogenase (SDHx)-mutated tumors has not been studied. Here, we evaluated the utility of SDHB/HIF-1α proteins in HNPGLs and PCCs/PGLs as clinically useful biomarkers. METHODS: The SDHB/succinate dehydrogenase subunit A (SDHA)/HIF-1α immunohistochemistry analysis was performed in 158 genetically defined patients. RESULTS: Similarly to PCCs/PGLs, SDHB immune-negativity correlated with SDHx-mutations in HNPGLs (P < .0001). The HIF-1α stabilization was associated with SDHx-mutations in HNPGLs (P = .020), not in PCCs/PGLs (P = .319). However, 25% of SDHx-HNPGLs lacked HIF-1α positive cells. CONCLUSION: As in PCCs/PGLs, SDHB immunohistochemistry in HNPGLs is a valuable method for identification of candidates for SDHx-genetic testing. On the contrary, although SDHx mutations may favor HIF-1α stabilization in HNPGLs, this is not a clinically useful biomarker.

Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate.

The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

Hyaluronic acid gel weight: a nonsurgical option for the management of paralytic lagophthalmos.

OBJECTIVES/HYPOTHESIS: Management of lagophthalmos should be a priority in the treatment of patients with facial palsy. The aim of the study was to evaluate the safety and efficacy of injecting hyaluronic acid gel into the upper eyelid as a nonsurgical alternative for patients with temporary facial palsy. METHOD/STUDY DESIGN: Retrospective study of 26 patients treated with hyaluronic acid gel injected into the pretarsal region of the upper eyelid. Measurements taken before and after treatment were standardized and compared using digitized photographs. Patients were followed up for 1 year, and overall outcomes were assessed. RESULTS: All patients initially demonstrated improvement in lagophthalmos, which decreased to 0.0 mm. After 1 month, a significant increase in lagophthalmos was observed in two patients (initial fissure of 8 and 9 mm), and a platinum weight was implanted to control keratopathy. The remaining patients (initial lagophthalmos below 6.5 mm) maintained the improvement until facial restoration. Only three patients had recurrent lagophthalmos (2 mm) due to resorption, which was resolved by injecting an additional 0.3 cc. The mean improvement in lagophthalmos was 4.6 mm (range, 3.5-6.5 mm). Complications included transient ecchymosis and minimal blepharoptosis due to nonreabsorption in five patients. These patients were successfully treated with hyaluronidase. CONCLUSIONS: Hyaluronic acid gel has proven effective in reducing paralytic lagophthalmos and controlling keratopathy in patients with temporary facial palsy, especially those with palpebral fissure with attempted closure no greater than 6.5 mm. Injection of hyaluronic acid gel is safe, quick, and easily performed. In addition, it is more cost-effective than surgery.

Identification of somatic VHL gene mutations in sporadic head and neck paragangliomas in association with activation of the HIF-1α/miR-210 signaling pathway.

CONTEXT: Head and neck paragangliomas (HNPGLs) arise from parasympathetic paraganglias and 35% to 45% are hereditary caused by mutations in succinate dehydrogenase (SDH) genes. The connection between SDH and tumor development is unclear. The most accepted hypothesis proposes a central role for the pseudohypoxic (pHx) pathway activated by hypoxia-inducible factor (HIF). Paradoxically, we showed that activation of HIF in HNPGLs is restricted to a subset of HNPGLs lacking SDH mutations. These tumors overexpress HIF-1α protein and target genes and the HIF-inducible microRNA miR-210 (pHx-HNPGLs). OBJECTIVE: The present study aimed at unraveling the SDH-independent mechanisms involved in the activation of HIF in HNPGLs. DESIGN: The VHL gene was analyzed in 53 tumors by gene sequencing, multiplex-ligation-dependent probe amplification, and quantitative PCR. The miR-210, HIF-1α, and CA9 levels were used as markers of the pHx gene signature. Meta-analysis of the transcriptome of pHx-HNPGLs was performed using the Oncomine platform. Assays in cells lacking functional pVHL and HIF-1α were performed to analyze the role of pVHL/HIF-1α on miR-210 expression. RESULTS: We identified, for the first time, somatic VHL mutations in HNPGLs. These were found in 2 of 4 pHx-HNPGLs with concomitant loss of heterozygosity in one of them; but not in non-pHx-HNPGLs. Meta-analysis of the transcriptome of pHx-HNPGLs revealed that these tumors are highly related to clear cell renal cell carcinoma. Cell-based assays showed that loss of pVHL lead to upregulation of miR-210 mainly via HIF-1α activation. CONCLUSIONS: VHL, involved in tumorigenesis of PGLs and clear cell renal cell carcinomas, may be an important player in the pathogenesis of sporadic HNPGLs via activation of an HIF-1α/miR-210 pHx pathway.

Identification of a signaling axis HIF-1α/microRNA-210/ISCU independent of SDH mutation that defines a subgroup of head and neck paragangliomas.

BACKGROUND: Head and neck paragangliomas (HNPGLs) are rare tumors associated with the parasympathetic nervous system. Most are sporadic, but about one third result from germline mutations in succinate dehydrogenase (SDH) genes (SDHB, SDHC, SDHD, SDHA, or SDHAF2). Although a molecular connection between SDH dysfunction and tumor development is still unclear, the most accepted hypothesis proposes a central role of the pseudohypoxic pathway. SDH dysfunction induces abnormal stabilization of the hypoxia-inducible factors (HIFs) that regulate target genes involved in proliferation, apoptosis, angiogenesis, and metabolism. The involvement of these pathways in the development of sporadic HNPGLs is presently unknown. OBJECTIVE: To get some insights into the hypoxic/pseudohypoxic molecular basis of HNPGLs, we attempted to define the gene, microRNA (miRNA), and HIF-1α expression patterns that distinguish tumors from normal paraganglia tissue. DESIGN: Genome microarray and TaqMan low-density arrays were used to analyze gene and miRNA expression, respectively, in 17 HNPGL tumor tissues and three normal human carotid bodies. Twelve HNPGLs were used for validation of data. HIF-1α, SDHB, and iron-sulfur cluster scaffold protein (ISCU) protein expression was analyzed by immunohistochemistry. RESULTS: We found activation of a canonical HIF-1α-related gene expression signaling only in a subset of HNPGLs from patients that did not harbor germline or somatic SDH mutations. The pseudohypoxic signature consisted in the overexpression of both HIF-1α-target genes and the HIF-1α-inducible miRNA, miR-210, and down-regulation of the miR-210 target gene, ISCU1/2. A decreased level of the iron-sulfur-containing protein SDHB was found by immunohistochemical analysis performed in two of these tumors. CONCLUSIONS: Collectively, this study unveiled a putative signaling axis of HIF-1α/miRNA-210/ISCU in a subset of HNPGLs that could have an impact on SDHB protein stability by a mechanism independent of SDH mutations, thus providing a foundation to better understand the functional interplay between HIF, miR-210, and mitochondria and its relevance in the pathogenesis of HNPGLs.

KRAS and BRAF mutations in sinonasal cancer.

OBJECTIVES: [corrected] Despite improvements in the field of surgery and radiotherapy, the overall prognosis of sinonasal carcinomas is poor, mainly due to the difficulty to resect the tumour completely in this anatomically complex region. Therefore, there is great need for alternative treatments. Knowledge of the KRAS and BRAF mutational status would become clinically important with regard to the possible use of anti-EGFR therapies. MATERIAL AND METHODS: DNA was extracted from paraffin embedded tumour samples from 57 cases of sinonasal squamous cell carcinoma (SNSCC) and from fresh frozen tumour samples from 58 cases of intestinal-type sinonasal adenocarcinoma (ITAC). Point mutations were analysed for KRAS exon 2 (codons 12 and 13) and BRAF (exon 15, V600E) by direct sequencing. RESULTS: Neither KRAS nor BRAF showed any mutations in the SNSCC, whereas 7/58 (12%) ITAC harboured KRAS mutations and no BRAF mutations. All seven cases with KRAS mutation concerned well-differentiated and less aggressive (papillary and colonic type) ITAC, all patients being woodworkers and 4/7 tobacco smokers. CONCLUSION: Neither of SNSCCs carried mutations in KRAS and BRAF and a low frequency of KRAS mutation was found in ITAC. This suggests that KRAS and BRAF mutations play a limited role in the development of sinonasal cancer and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in sinonasal cancer.

Gene amplification and protein overexpression of EGFR and ERBB2 in sinonasal squamous cell carcinoma.

BACKGROUND: Sinonasal squamous cell carcinomas (SNSCCs) are rare tumors with no etiologic link to tobacco or alcohol, as opposed to other squamous cell carcinomas of the head and neck. Despite improvements in the field of surgery and radiotherapy, patients with these tumors still face a very unfavorable prognosis, partly because of their localization in a complex anatomic area, which has special relevance for surgery and postoperative treatment. Therefore, there is a need for new therapeutic possibilities for patients with these tumors. METHODS: Gene copy numbers of epidermal growth factor receptor (EGFR) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2) were analyzed by fluorescence in situ hybridization and multiplex ligand-dependent probe amplification, and protein expression was evaluated by immunohistochemistry in 54 SNSCC specimens. The results were correlated with clinicopathologic and follow-up data. RESULTS: EGFR gene copy number increases were observed in 20 of 45 tumors (44%), and 21 of 54 tumors (39%) had EGFR protein overexpression. Eight of 38 tumors (21%) had ERBB2 copy number increases, and 4 of 54 tumors (7%) exhibited elevated protein expression levels. Both copy number increases and protein overexpression of EGFR and ERBB2 were mutually exclusive. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were absent in 37 tumors that were analyzed. CONCLUSIONS: A substantial proportion of SNSCCs carried alterations in EGFR or ERBB2. Together with the absence of KRAS mutations, these findings indicate that therapies targeting these molecules may be promising additions to the therapeutic options for patients with SNSCC.

Functional role of human laryngeal nerve connections.

OBJECTIVES/HYPOTHESIS: Current knowledge of the functional role of human laryngeal nerves is based on traditional laryngeal neuroanatomic descriptions or contradictory electromyographic studies. The aim of this study was to clarify the functional role of neural connections between laryngeal nerves by correlating the different electromyographic patterns observed after laryngeal stimulation and the existence of different neural connections. STUDY DESIGN: Descriptive. METHODS: Electromyographic and morphologic study in 13 patients during total laryngectomy procedure. RESULTS: Seven patients showed an additional evoked response from the cricothyroid muscle after recurrent laryngeal nerve stimulation. External laryngeal nerve stimulation resulted in additional responses from the posterior cricoarytenoid muscle in three cases and from the arytenoid muscle in one. The presence of a neural connection was confirmed in all patients who showed an unexpected electromyographic response. CONCLUSIONS: The different connections between laryngeal nerves are at least partially of motor nature and play a role in the mobility of vocal folds.

[Otorhinolaryngology]. / Otorrinolaringología.

The terrorist attack on 11 March caused a high percentage of patients with ear injuries. Shock waves provoke alterations to the external ear as well as to the middle and inner ear. The most frequent lesion is tympanic membrane perforation. Initial evaluation with otoscopy, acoumetry, tonal audiometry and vestibular examination was performed and was repeated after 2 and 3 months. In most patients there was a correlation between the grade of middle ear lesion and auditory damage. In most patients with tympanic rupture, the perforation was total or subtotal. Spontaneous closure can occur in some patients but is usually related to the size of the initial lesion. Thus in tympanic perforations of more than 50%, spontaneous closure is unlikely.

Otorrinolaringología / Otorhinolaryngology

El atentado del 11 de marzo ocasionó en un alto porcentaje de pacientes lesiones otológicas. La onda expansiva provoca alteraciones tanto del oído externo como del oído medio e interno. La perforación timpánica es la más frecuente de las consecuencias. En nuestro caso, realizamos un primer estudio con otoscopia, acumetría, audiometría tonal y exploración vestibular, estudio que se repitió a los 2 y 3 meses. En la mayoría de los casos se comprobó una correlación entre el grado de lesión del oído medio y el daño auditivo. De los pacientes con rotura timpánica, la mayor parte presentaban perforación total o subtotal. Un determinado porcentaje de casos puede cerrar espontáneamente, pero suele estar en relación con el tamaño de la lesión inicial. Así, en perforaciones timpánicas de más del 50% es difícil que se produzca un cierre espontáneo (AU)

The terrorist attack on 11 March caused a high percentage of patients with ear injuries. Shock waves provoke alterations to the external ear as well as to the middle and inner ear. The most frequent lesion is tympanic membrane perforation. Initial evaluation with otoscopy, acoumetry, tonal audiometry and vestibular examination was performed and was repeated after 2 and 3 months. In most patients there was a correlation between the grade of middle ear lesion and auditory damage. In most patients with tympanic rupture, the perforation was total or subtotal. Spontaneous closure can occur in some patients but is usually related to the size of the initial lesion. Thus in tympanic perforations of more than 50%, spontaneous closure is unlikely (AU)