Pyrosequencing-based DNA methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma

Fanconi anemia (FA) associated genes [FANCA, -B, -C, FANCD1(BRCA2), -D2, -E, -F, -G, -I, -L, -M, FANCN (PALB2), FANCJ(BRIP1) and FA-linked BRCA1] encode proteins of DNA damage response pathways mutated in FA patients. FA is characterized by congenital malformations, chromosomal instability and high cancer susceptibility. FA patients have a 500-700 times higher risk of head and neck squamous cell carcinoma (HNSCC) compared to the non-FA population. As DNA methylation comprises one of the known gene inactivation mechanisms in cancer we have investigated the methylation status of 13 FA and one FA-linked gene in order to assess the role of FA in sporadic laryngeal squamous cell carcinoma (LSCC) tumor samples. Thirteen laryngeal squamous carcinoma cell lines (UT-SCC) and 64 primary laryngeal carcinoma cases were analyzed by bisulfite pyrosequencing. DNA from buccal swabs of 10 healthy volunteers was used as a control group. Promoter regions of FANCA, BRCA1 and BRCA2 displayed recurrent alterations in the methylation levels in cancer samples as compared to buccal swabs controls. For FANCA, hypomethylation was observed in 11/13 cell lines (p<0.0003) and all 64 primary larynx samples (p<0.001) compared to buccal swabs. For BRCA1, 4/13 cell lines (p=0.04) and 3/58 primary laryngeal cases (p=0.22) showed hypomethylation. In BRCA2, all 13 cell lines (p<0.0001) 4/63 primary LSCC (p<0.01) showed hypermethylation as compared to controls. In conclusion, we show recurrent alterations of DNA methylation levels in three Fanconi anemia genes which might contribute to the pathogenesis of LSCC.     

p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells

Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.     

Tension‐free vaginal tape procedure without preoperative urodynamic examination: Long‐term outcome

Objectives: To evaluate the long‐term outcome of the tension‐free vaginal tape procedure. Methods: A total of 191 patients were operated on with tension‐free vaginal tape between January 1998 and May 2000. Of these, 127 (66%) had stress urinary incontinence, 64 (34%) had mixed urinary incontinence and 39 (20%) had recurrent incontinence. A total of 34 (18%) patients had had concomitant surgery. The diagnosis of incontinence was based on a history of leakage during stress and physical examination with a supine stress test in all patients. Tension‐free vaginal tape was carried out under local (82%) or spinal (18%) anesthesia. After a mean of 10.5 years follow up, the assessment included a gynecological examination and a supine stress test. Subjective outcome was evaluated with Urinary Incontinence Severity Score, Detrusor Instability Score, visual analog scale, European quality of life‐five dimensions, European quality of life – visual analog scale and short versions of Incontinence Impact Questionnaire‐7 and Urogenital Distress Inventory‐6. Objective cure was defined as a negative stress test and an absence of reoperation for incontinence during the follow up. Results: A total of 138 (72%) of 191 patients were evaluated. Patients with minimally invasive surgery before operation had significantly higher scores in Urinary Incontinence Severity Score, Detrusor Instability Score, Incontinence Impact Questionnaire‐7 and Urogenital Distress Inventory‐6 at follow up than the patients with stress urinary incontinence (P < 0.01). Recurrent incontinence and concomitant surgery did not affect the long‐term outcome. Three patients (2.3%) had late‐onset adverse events. The objective and subjective cure rates were 90% and 78%, respectively. Conclusions: The tension‐free vaginal tape procedure is effective and safe even after 10 years. The objective cure rate is high, but the subjective outcome is significantly lower in mixed urinary incontinence patients compared with patients with pure stress urinary incontinence. Recurrent stress urinary incontinence does not affect the outcome, and tape‐related problems are rare.     

Oral Mucosa as a Reservoir of Human Papillomavirus: Point Prevalence,Genotype Distribution,and Incident Infections Among Males in a 7-year Prospective Study

Background

In addition to the anogenital malignancies, human papillomavirus (HPV) has been linked to oropharyngeal cancer as an important risk factor in both men and women. Knowledge of oral HPV infection among males is needed to elucidate the transmission routes and potential for prevention.

Objective

To assess the prevalence, genotype distribution, and incidence of oral HPV infections among healthy Finnish men followed for 7 yr.

Design, setting, and participants

Oral scrapings for HPV testing were taken from 131 fathers-to-be (mean age: 28.9 yr) at baseline and at 2-mo, 6-mo, 12-mo, 24-mo, 36-mo, and 7-yr follow-up visits to detect prevalent and incident HPV infections. Purified DNA extracted from scrapings was used for HPV genotyping, with the Multimetrix kit (Progen Biotechnik, Heidelberg, Germany) detecting 24 genotypes.

Outcome measurements and statistical analysis

Point prevalence, genotype distribution, and incident rates of oral HPV infections. Demographic data were collected using structured questionnaires, and covariates of incident oral HPV infections were analysed using uni- and multivariate Poisson regression (for panel data).

Results and limitations

The point prevalence of oral HPV infection fluctuated from 15.1% to 31.1% during the follow-up period. In total, 17 different HPV genotypes were found. At baseline, the single most frequent genotype among the HPV-positive samples was HPV16 (33.3%; 8 of 24), followed by HPV33 (12.5%) and HPV82 (12.5%). Multiple-type infections comprised 16.7% (4 of 24), HPV16 being involved in all combinations. For baseline-negative men, the mean time to the first incident infection ranged from 3.9 mo (HPV82) to 25.7 mo (HPV56). None of the demographic factors was a significant independent predictor of incident oral HPV infections in multivariate models.

Conclusions

Detection of oral HPV DNA carriage in men is common, HPV16 being the most prevalent genotype. Oral mucosa may play a significant role in HPV transmission.     

Complete avulsion of the splenic vascular pedicle as a birth injury

The survivor of a rare birth injury is reported. The patient was a term girl weighing 5,340 g at birth. At the age of 2 days abdominal symptoms began (vomiting and distenison). At laparotomy at 4 days of age, the spleen was found completely avulsed from its vascular pedicle and was removed. The abdomen was full of blood, but there was no acute bleeding. The postoperative course was uneventful. At re-examination at the age of 3 months she was in good health. To minimize the risk of overwhelming sepsis due to encapsulated bacteria, vaccinations are in progress. Offprint requests to: H. Lindahl     

Acquired pure red-cell aplasia: A consequence of increased natural killer cell activity?

A 19-year-old man with severe pure red-cell aplasia is described. An unusually high proportion of this patient's lymphocytes were large granular lymphocytes (LGL), both in the blood (40%) and in the bone marrow (50%). His blood leukocytes displayed a strongly elevated natural killer (NK) cell activity in vitro against the erythroblastic leukemia line K562. The patient's non-T blood lymphocytes inhibited in vitro erythroid colony formation (BFU-E and CFU-E) but not the granulocyte-monocyte colony growth (CFU-GM) from autologous and allogeneic bone marrow. Neither T-cell-mediated cytotoxicity nor circulating antibodies against erythroid precursors could be demonstrated. The patient's haemoglobin values returned to normal levels after three weeks of glucocorticoid treatment and have since then remained stable with continued prednisone administration. Attempts to reduce the prednisone dose to less than 10 mg/day have led to relapses. It is tempting to suggest that the patient's disease might be caused by hyperactivity of cytotoxic non-T (NK) cells specific for K562 cells and early erythroid precursors.     

Recent life changes and coronary heart disease in East versus West Finland

Coronary heart disease (CHD) is not equally prevalent in East vs West Finland. Substantially higher CHD rates are found in East Finland, and these higher rates are only partially explained by differences in physiological “risk factors” such as body mass, blood pressure, cigarette smoking, and serum cholesterol levels between persons in these two regions. Recent life changes magnitudes were assessed for representative groups of men living in these two areas of Finland. East Finns reported approximately 84% higher life change magnitude than West Finns. Subjects' life changes magnitudes were essentially unrelated to their physiological risk factors. The relatively increased life change levels seen for East Finns may constitute an environmental risk factor for CHD.