Role of Epstein-Barr virus DNA measurement in plasma in the clinical management of nasopharyngeal carcinoma in a low risk area.

OBJECTIVE: To evaluate the role of quantitative measurement of Epstein-Barr virus (EBV) DNA in the clinical management of nasopharyngeal carcinoma (NPC) in a low tumour risk area (western Europe). METHODS: 22 consecutive Dutch NPC patients (11 europid) were studied. EBV DNA load in pretreatment and post-treatment plasma samples was determined. Three patients were also sampled at frequent intervals during treatment. RNA in situ hybridisation for the detection of EBV encoded RNAs (EBERs) was carried out on tumour biopsies of all cases. RESULTS: All patients with EBER positive NPC (20/22) showed a positive EBV DNA load in plasma at the time of diagnosis (median EBV DNA level, 4.1 log(10) copies/ml). Patients with EBER negative NPC had no detectable EBV DNA in plasma. After treatment, complete remission was achieved in all cases and concurrently EBV DNA in plasma became undetectable in all patients. In the three longitudinally evaluated cases, EBV DNA load gradually declined towards undetectable levels within three weeks after start of treatment. Two patients developed a distant metastasis with concomitant increases in EBV viral load. In addition, one EBER positive patient developed an EBER negative metastasis in the neck during follow up and in this case EBV DNA load remained undetectable at the time of recurrence. CONCLUSIONS: Plasma EBV DNA load measurement appears to be useful in a low tumour risk area. However, development of local recurrences may not always coincide with raised levels of EBV DNA.

Increased prevalence of catecholamine excess and phaeochromocytomas in a well-defined Dutch population with SDHD-linked head and neck paragangliomas.

OBJECTIVE: The aim of this study was to identify the prevalence of catecholamine excess and phaeochromocytomas in a well-defined population of people with hereditary head and neck paragangliomas. METHODS: We studied in a prospective follow-up protocol all consecutive patients referred to the Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands with documented head and neck paragangliomas and either a positive family history for paragangliomas or a proven SDHD gene mutation. Initial analysis included medical history, physical examination and the measurement of excretion of catecholamines in two 24-h urine collections. In the case of documented catecholamine excess iodinated meta-iodobenzylguanidine (123I-MIBG) scintigraphy and magnetic resonance imaging were done. RESULTS: Between 1988 and 2003, 40 consecutive patients (20 male and 20 female) with documented head and neck paragangliomas were screened. Biochemical screening revealed urinary catecholamine excess in 15 patients (37.5%). In nine of these 15 patients a lesion was found by 123I-MIBG scintigraphy. Exact localization by magnetic resonance imaging revealed phaeochromocytomas in seven of the 15 patients. One of the nine patients had an extra-adrenal paraganglioma. Histopathological examination in a subset of tumors displayed loss of heterozygosity of the wild-type SDHD allele in all cases. CONCLUSIONS: The prevalence of catecholamine excess (37.5%) and phaeochromocytomas (20.0%) is high in patients with familial head and neck paragangliomas. Therefore, patients with hereditary head and neck paragangliomas require lifelong follow up by biochemical testing for catecholamine excess.

SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology.

Hereditary head and neck paragangliomas are tumours associated with the autonomic nervous system. Recently, mutations in genes coding for subunits of mitochondrial complex II, succinate-ubiquinone-oxidoreductase (SDHB, SDHC, and SDHD), have been identified in the majority of hereditary tumours and a number of isolated cases. In addition, a fourth locus, PGL2, has been mapped to chromosome 11q13 in an isolated family. In order to characterize phenotypic effects of these mutations, the present study investigated the immunohistochemical expression of the catalytic subunits of complex II (flavoprotein and iron protein), SDH enzyme activity, and mitochondrial morphology in a series of 22 head and neck paragangliomas. These included 11 SDHD-, one SDHB-, two PGL2-linked tumours, and eight sporadic tumours. In the majority of the tumours (approximately 90%), the enzyme-histochemical SDH reaction was negative and immunohistochemistry of catalytic subunits of complex II showed reduced expression of iron protein and enhanced expression of flavoprotein. Ultrastructural examination revealed elevated numbers of tightly packed mitochondria with abnormal morphology in SDHD-linked and sporadic tumours. Immuno-electron microscopy showed localization of the flavoprotein on the remnants of the mitochondrial inner membranes, whereas virtually no signal for the iron protein was detected. These results indicate that the function of mitochondrial complex II is compromised in the majority of head and neck paragangliomas.

Microwave immunoincubations using coverplate units.

Recently, the Coverplate immunostaining method was introduced. This system allows easy handling of the slides and is suitable for immunoincubations for a variety of antigens at the same time on consecutive sections, without having to apply droplets of the individual primary antibodies on the sections. In this study, temperature rise inside the Coverplate units during microwave exposure is investigated. The most suitable conditions for microwave immunoincubations are determined. Immunoincubations with the Coverplate unit are conducted in the microwave oven and the results evaluated.

Effect of aluminum chloride, -citrate, and -maltol on the calcium-mediated degradation of neurofilament proteins.

Aluminum (Al) has been observed to cause neurofilament protein accumulation in both experimental animals and cultured cells. Impairment of axonal transport is thought to be a mechanism of toxicity. Inhibition of the degradation of neurofilament proteins, however, resulting in accumulation of these proteins may be an alternative mechanism for Al toxicity. In the present study, the effect of calcium (Ca) on the proteolysis of the neurofilament triplet proteins by calcium-activated neutral proteases (CANP) was studied in the isolated sciatic nerve explants. The extent of the degradation was found to be dependent on the Ca concentration. The effect of Al chloride, -citrate and -maltol on the calcium-induced degradation was studied. No effect of any of the Al compounds was observed, suggesting that the metal may exert its neurotoxic effect via a mechanism other than impairment of neurofilament proteolysis. Maltol itself was found to enhance the effect of Ca on the degradation of neurofilament proteins, probably by facilitating the movement of Ca across the neuronal membrane.