63. Raki Płaskonabłonkowe głowy i szyi u młodych dorosłych – retrospektywna wieloczynnikowa analiza oraz wstępne wyniki badań nad rolą czynnika genetycznego w ich etiopatogenezie

Celem pracy jest analiza cech klinicznych raków płaskonabłonkowych głowy i szyi u chorych poniżej 45 roku życia („young adult”) oraz ocena udziału czynnika genetycznego w ich etiopatogenezie. W ocenie klinicznej uwzględniono dane dotyczące: A.- pacjentów: wiek (średnia 40.9 lat), płeć (mężczyźni 85%, kobiety 15%) oraz ekspozycja na typowe czynniki ryzyka (palący papierosy −96.8%, spożywający alkohol −96.7%), B.- guza: lokalizacja (krtań 69%, jama ustna i gardło 28%), stopień zaawansowania klinicznego (głównie stadium IV −58.7%), typ histologiczny (ca planoepitheliale keratodes 71.2%, akeratodes 28.8%) i stopień zróżnicowania histologicznego (głownie G1+G2 −94.3%), C.- zastosowanego leczenia: metoda (głównie skojarzone: operacja + radioterapia −61.3%) oraz wyniki (wznowa miejscowa −15.9%, wznowa węzłowa −18.5%, przeżycia 3-letnie 46%, przeżycia 5-letnie 35%). W ocenie roli czynnika genetycznego zastosowano 3 techniki: genotypowanie, test bleomycynowy i test kometkowy. U 24 chorych oznaczono polimorfizm genów metabolizmu kancerogenów oraz genów związanych z naprawą DNA. Stwierdzono częstsze niż w grupie pacjentów starszych występowanie następujących genotypów ryzyka: GSTM1 (-) współwystępujący z NAT2 4/6A oraz NAT2 4/6A oraz allelu ryzyka: NAT2 4; allel ryzyka CYP 1A1 4 występował rzadziej niż w grupie starszych, natomiast częstość występowania genotypu ryzyka XPD 35931AA w obu grupach była zbliżona. U 5 pacjentów oznaczono poziom niestabilności chromosomowej za pomocą testu bleomycynowego – stwierdzono zbliżone wartości współczynnika b/c w grupie pacjentów młodszych i starszych przy jednocześnie wyraźnie większym odsetku uszkodzonych komórek w grupie młodych dorosłych. W obu grupach zarówno wartość współczynnika b/c jak i procent uszkodzonych komórek były wyrażnie większe niż w grupie osób zdrowych. W grupie 7 pacjentów za pomocą techniki comet assay określono uszkodzenia spontaniczne i indukowane oraz zdolności naprawy DNA – stwierdzono wysoki i zbliżony w grupie pacjentów młodych i starszych poziom uszkodzeń DNA – zarówno spontanicznych i indukowanych oraz zbliżone zdolności naprawy DNA

Copy Number Gains of the Putative CRKL Oncogene in Laryngeal Squamous Cell Carcinoma Result in Strong Nuclear Expression of the Protein and Influence Cell Proliferation and Migration

Laryngeal squamous cell carcinoma is a major medical problem worldwide. Although our understanding of genetic changes and their consequences in laryngeal cancer has opened new therapeutic pathways over the years, the diagnostic as well as treatment options still need to be improved. In our previous study, we identifed CRKL (22q11) as a novel putative oncogene overexpressed and amplifed in a subset of LSCC tumors and cell lines. Here we analyze to what extent CRKL DNA copy number gains correlate with the higher expression of CRKL protein by performing IHC staining of the respective protein in LSCC cell lines (n=3) and primary tumors (n=40). Moreover, the importance of CRKL gene in regard to proliferation and motility of LSCC cells was analyzed with the application of RNA interference (siRNA). Beside the physiological cytoplasmic expression, the analysis of LSCC tumor samples revealed also nuclear expression of CRKL protein in 10/40 (25%) cases, of which three (7.5%), presented moderate or strong nuclear expression. Similarly, we observed a shift towards aberrantly strong nuclear abundance of the CRKL protein in LSCC cell lines with gene copy number amplifcations. Moreover, siRNA mediated silencing of CRKL gene in the cell lines showing its overexpression, signifcantly reduced proliferation (p<0.01) as well as cell migration (p<0.05) rates. Altogether, these results show that the aberrantly strong nuclear localization of CRKL is a seldom but recurrent phenomenon in LSCC resulting from the increased DNA copy number and overexpression of the gene. Moreover, functional analyses suggest that proliferation and migration of the tumor cells depend on CRKL expression.</p

Combined deletion and DNA methylation result in silencing of FAM107A gene in laryngeal tumors

Larynx squamous cell carcinoma (LSCC) is characterized by complex genotypes, with numerous abnormalities in various genes. Despite the progress in diagnosis and treatment of this disease, 5-year survival rates remain unsatisfactory. Therefore, the extended studies are conducted, with the aim to find genes, potentially implicated in this cancer. In this study, we focus on the FAM107A (3p14.3) gene, since we found its significantly reduced expression in LSCC by microarray profiling (Affymetrix U133 Plus 2.0 array). By RT-PCR we have confirmed complete FAM107A downregulation in laryngeal cancer cell lines (15/15) and primary tumors (21/21) and this finding was further supported by FAM107A protein immunohistochemistry (15/15). We further demonstrate that a combined two hit mechanism including loss of 3p and hypermethylation of FAM107A promoter region (in 9/15 cell lines (p p FAM107A expression (5 to 6 fold increase) in the UT-SCC-29 cell line, characterized by high DNA methylation. Therefore, we report the recurrent inactivation of FAM107A in LSCC, what may suggest that the gene is a promising tumor suppressor candidate involved in LSCC development.</p

Recurrent epigenetic silencing of the PTPRD tumor suppressor in laryngeal squamous cell carcinoma

Cellular processes like differentiation, mitotic cycle, and cell growth are regulated by tyrosine kinases with known oncogenic potential and tyrosine phosphatases that downmodulate the first. Therefore, tyrosine phosphatases are recurrent targets of gene alterations in human carcinomas. We and others suggested recently a tumor suppressor function of the PTPRD tyrosine phosphatase and reported homozygous deletions of the PTPRD locus in laryngeal squamous cell carcinoma. In this study, we investigated other gene-inactivating mechanisms potentially targeting PTPRD, including loss-of-function mutations and also epigenetic alterations like promoter DNA hypermethylation. We sequenced the PTPRD gene in eight laryngeal squamous cell carcinoma cell lines but did not identify any inactivating mutations. In contrast, by bisulfite pyrosequencing of the gene promoter region, we identified significantly higher levels of methylation (p = 0.001 and p = 0.0002, respectively) in 9/14 (64%) laryngeal squamous cell carcinoma cell lines and 37/79 (47%) of primary laryngeal squamous cell carcinoma tumors as compared to normal epithelium of the upper aerodigestive tract. There was also a strong correlation (p = 0.0001) between methylation and transcriptional silencing for the PTPRD gene observed in a cohort of 497 head and neck tumors from The Cancer Genome Atlas dataset suggesting that DNA methylation is the main mechanism of PTPRD silencing in these tumors. In summary, our data provide further evidence of the high incidence of PTPRD inactivation in laryngeal squamous cell carcinoma. We suggest that deletions and loss-of-function mutations are responsible for PTPRD loss only in a fraction of cases, whereas DNA methylation is the dominating mechanism of PTPRD inactivation.</p

Inventory of current EU paediatric vision and hearing screening programmes

Background: We examined the diversity in paediatric vision and hearing screening programmes in Europe. Methods: Themes relevant for comparison of screening programmes were derived from literature and used to compile three questionnaires on vision, hearing and public-health screening. Tests used, professions involved, age and frequency of testing seem to influence sensitivity, specificity and costs most. Questionnaires were sent to ophthalmologists, orthoptists, otolaryngologists and audiologists involved in paediatric screening in all EU fullmember, candidate and associate states. Answers were cross-checked. Results: Thirty-nine countries participated; 35 have a vision screening programme, 33 a nation-wide neonatal hearing screening programme. Visual acuity (VA) is measured in 35 countries, in 71% more than once. First measurement of VA varies from three to seven years of age, but is usually before the age of five. At age three and four picture charts, including Lea Hyvarinen are used most, in children over four Tumbling-E and Snellen. As first hearing screening test otoacoustic emission (OAE) is used most in healthy neonates, and auditory brainstem response (ABR) in premature newborns. The majority of hearing testing programmes are staged; children are referred after one to four abnormal tests. Vision screening is performed mostly by paediatricians, ophthalmologists or nurses. Funding is mostly by health insurance or state. Coverage was reported as >95% in half of countries, but reporting was often not first-hand. Conclusion: Largest differences were found in VA charts used (12), professions involved in vision screening (10), number of hearing screening tests before referral (1-4) and funding sources (8)