Xavier León

Second neoplasm in patients with head and neck cancer

The improvement in locoregional control of head and neck carcinomas over the last decades does not appear to modify the final survival of these patients, mainly due to the appearance of distant metastases and second neoplasms. We ran a study to evaluate the incidence of second neoplasms and their characteristics in patients with head and neck carcinoma treated in our hospital.

Distant metastases in head and neck cancer patients who achieved loco-regional control.

One of the reasons for failure in patients with head and neck carcinoma who achieve locoregional control with treatment is the appearance of distant metastases. The objective of this study was to evaluate the frequency of distant metastases in this group of patients and to determine the relative role of several prognostic factors in the subsequent development of distant metastases.

HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients

BACKGROUND We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation. METHODS Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results. RESULTS A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America. CONCLUSIONS HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs.

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency.

The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, second-generation CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.

An anatomical study of anastomoses between the laryngeal nerves

Objective: To systematize the anatomy of the connecting branches between laryngeal nerves. Methods: Microdissection of 90 larynges obtained from necropsies (57 men and 33 women; age range, 41–95 y). Results: Anastomoses between the internal and recurrent nerves appeared in four different patterns: 1) Galens anastomosis, as the connection between the dorsal branches of both nerves (100%); 2) arytenoid plexus, as the connection between the arytenoid branches of both nerves, in relation with the arytenoid muscle, and divided in a deep part (100%) and a superficial part (86%); 3) cricoid anastomosis, previously only described in cows, located in the front of the cricoid lamina (6/10 cases); and 4) thyroarytenoid anastomosis, as the connection of a descending branch of the internal laryngeal nerve and an ascending branch of the recurrent nerve (14%). Anastomosis between the internal laryngeal and the external laryngeal nerves appeared as a connecting branch throughout the foramen thyroideum (21%). Anastomosis between the external laryngeal and recurrent nerves appeared as a connecting branch throughout the cricothyroid muscle (68%). Conclusion: At least two anastomoses (Galens anastomosis and arytenoid plexus) appeared in 21% of hemilarynges, and 79% of cases had three or more anastomoses between the laryngeal nerves. The different prevalence of this complex anastomotic pattern suggests functional differences in the sensory and motor innervation of individual subjects. Key Words: Larynx, innervation, anastomosis, nerves.

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

Second Primary Tumors in Head and Neck Cancer Patients

XAVIER LEÓN, ALFIO FERLITO, CHARLES M. MYER III, UMBERTO SAFFIOTTI, ASHOK R. SHAHA, PATRICK J. BRADLEY, MARGARET S. BRANDWEIN, MATTI ANNIKO, RAVINDHRA G. ELLURU and ALESSANDRA RINALDO From the Department of Otolaryngology, Hospital de Santa Creu i Sant Pau, Barcelona, Spain, Department of Otolaryngology—Head and Neck Surgery, University of Udine, Udine, Italy, Department of Pediatric Otolaryngology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland , USA, Head and Neck Service, Memorial Sloan–Kettering Cancer Center, New York, New York, USA, Department of Otorhinolaryngology —Head and Neck Surgery, Queen’s Medical Centre, Nottingham, UK, Department of Otolaryngology and Pathology, Mount Sinai School of Medicine, New York, New York, USA and Department of Otolaryngology—Head and Neck Surgery, University Hospital, Uppsala, Sweden

Is the external laryngeal nerve an exclusively motor nerve? The cricothyroid connection branch

Objectives To obtain an accurate morphological description of the nerve that provides communication between the external laryngeal nerve and the recurrent laryngeal nerve in a large sample of human larynges.

Influence of the persistence of tobacco and alcohol use in the appearance of second neoplasm in patients with a head and neck cancer. A case-control study

ObjectiveTo evaluate the influence of persistent tobacco and alcohol use on the risk of a second metachronous neoplasm in the aerodigestive tract in head and neck squamous cell carcinoma (HNSCC) patients.MethodsA matched case–control study was carried out in 514 patients with HNSCC. Case patients developed a second metachronous neoplasm in the aerodigestive tract after treatment of an index HNSCC. A patient free of second neoplasm was individually matched to every case patient by location of the index tumor, tumor stage, sex, previous tobacco and alcohol consumption, age, general health status, and treatment. Data about persistence in tobacco and alcohol consumption after treatment of the index tumor was collected retrospectively. A validation study was carried out to confirm the adequacy of this retrospective information.ResultsPersistent tobacco smoking and alcohol drinking after treatment of a HNSCC contributed to the risk of appearance of second neoplasm. The odds ratio of a second neoplasm for patients who continued to smoke was 2.9 (95% CI OR 1.8–4.1), and for patients who continued to use alcohol it was 5.2 (95% CI OR 3.3–7.9). There was a strong association between persistence of tobacco and alcohol use after treatment of the HNSCC index tumor. According to the attributable risk estimation, persistent tobacco and alcohol consumption would be responsible for one-third of the second neoplasms in the patients with a HNSCC index tumor.ConclusionsPersistence of tobacco and alcohol use after treatment of a HNSCC had a significant influence on the appearance of a second neoplasm in the aerodigestive tract. Cessation of tobacco and alcohol use should be a major goal after treatment of a HNSCC.

Prevention of stomal recurrence.

The authors reviewed the incidence of stomal recurrence (SR) in a population of laryngectomized patients to study associated risk factors and determine the usefulness of certain preventive procedures.