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MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3′UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.

Genomic and mitochondrial DNA molecules are exposed continuously for a damaging activity of chemical, physical, and internal genotoxicants. When DNA repair machinery is not working efficiently, the generation of DNA lesions and mutations leads to carcinogenic transformation. The high number of mutation going up to 105 per cell was recognized as a driving force of oncogenesis. Moreover, a high activity of DNA repair genes was hypothesized as a predisposition to metastasis. DNA repair potential has to be taken into account attempting to chemo- and/or radiotherapy. A low activity of DNA repair genes makes tumor cells more sensitive to therapy, but on the other hand, non-tumor cells getting lesions could form second primary cancer. Contrary, high activity of DNA repair genes counteracts attempted therapy. It means an individualized therapy based on recognition of DNA repair potential is recommended.

Purpose of review Observational studies have shown that serum 25-OH vitamin D [25(OH)D] is inversely associated with overall cancer risk in many malignancies. We performed a systematic literature review to determine whether vitamin D deficiency is related to head and neck cancer (HNC) etiology and outcome. Recent findings The search yielded five prospective studies reporting 25(OH)D levels prior to cancer diagnosis and their effect on the risk of HNC. Eight studies were cross-sectional or case-control studies, in which 25(OH)D levels were only measured after cancer diagnosis. Two studies found an inverse association between 25(OH)D level and HNC risk, while two other prospective cohort studies demonstrated no connection between 25(OH)D and HNC risk. Several studies reported cancer patients to have significantly lower 25(OH)D levels than controls. Associations between 25(OH)D and prognosis and mortality were variable. Summary The link between vitamin D and HNC has so far only been investigated in a few observational, prospective, and case-control studies. Vitamin D deficiency may be more common in HNC patients than in the healthy population. There is no evidence for a causal relationship. Further studies are needed to evaluate whether low 25(OH)D concentrations play a role in the development or outcome of HNCs.

Simple Summary The risk of developing cancer is always higher for tobacco smokers than for non-smokers. Electronic cigarettes (e-cigarettes) have become increasingly popular in the last decade and are considered less harmful than traditional tobacco products, due to the lower content of toxic and carcinogenic compounds. However, this is still a controversial issue. This paper contains a review of previous reports on the composition of e-cigarettes and their impact on the pathogenesis and risk of head and neck cancer (HNC). The authors reviewed articles on both toxic and carcinogenic compounds contained in e-cigarettes and their molecular and health effects on the upper respiratory tract in comparison to traditional tobacco cigarettes. In conclusion, the studies discussed in the review strongly suggest that more long-term studies are needed to better address the safety of e-cigarettes. Abstract E-cigarettes have become increasingly popular in the last decade and are considered less harmful than traditional tobacco products due to the lower content of toxic and carcinogenic compounds. However, this is still a controversial issue. This paper contains a review of previous reports on the composition of e-cigarettes and their impact on the pathogenesis and risk of head and neck cancer (HNC). The objective of the review was to compare the molecular and health effects of e-cigarette use in relation to the effects of traditional cigarette smoking in the upper respiratory tract, and to assess the safety and effect of e-cigarettes on HNC risk. A review for English language articles published until 31 August 2020 was made, using a PubMed (including MEDLINE), CINAHL Plus, Embase, Cochrane Library and Web of Science data. The authors reviewed articles on both toxic and carcinogenic compounds contained in e-cigarettes and their molecular and health effects on the upper respiratory tract in comparison to tobacco cigarettes. The risk of developing head and neck squamous cell carcinoma (HNSCC) remains lower in users of e-cigarettes compared with tobacco smokers. However, more long-term studies are needed to better address the safety of e-cigarettes.

There is an agreement about joint genetic and environmental background of musical reception and performance. Musical abilities tend to cluster in families. The studies done on a random population, twins and families of gifted musicians provided a strong support for genetic contribution. Modern biomolecular techniques exploring linkage analysis, variation of gene copy number, scanning for whole-genome expression helped to identify genes, or chromosome regions associated with musical aptitude. Some studies were focused on rare ability to recognize tone without reference that is known as a perfect pitch where a far ethnic differentiation was established. On the other hand, gene deletion leading to dysfunction in amusical individuals also indicated appropriate loci “by negation.” The strongest support for an association of genes with musicality was provided for genes: AVPR1 (12q14.2), SLC6A4 (17q11.2), GALM (2p22), PCDH7 (4p15.1), GATA2 (3q21.3), and few others as well for 4q22, 4q23, and 8q13–21 chromosome bands.

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 identified CRKL (22q11) as a novel putative oncogene overexpressed and amplified 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 amplifications. Moreover, siRNA mediated silencing of CRKL gene in the cell lines showing its overexpression, significantly reduced proliferation (p CRKL expression.

Item does not contain fulltext The objective of the review was to compare molecular and health effects of tobacco smoking using cigars, cigarillos, pipe and water pipe in relation to the effects of cigarette smoking. In this review we will focus on the upper respiratory tract. Mechanisms of interaction of tobacco smoke constituents after products other than cigarettes are similar to these associated with cigarette smoking. Carcinogenic activity was demonstrated for any type of tobacco smoking, although the risk of developing head and neck squamous cell carcinoma (HNSCC) remains lower in users of cigars, traditional pipe and water pipe as compared to cigarette smoking. Nevertheless, there is no way of safe tobacco smoking.

Meniere’s disease (MD) as an inner ear disorder including such symptoms as recurrent vertigo attacks, tinnitus, fluctuating or progressive sensorineural hearing loss. Its relatively frequent familial incidence implicates a genetic background. An autosomal dominant inheritance was commonly observed with a few exceptions. It was established that Meniere’s disease is not a monogenic disorder. Instead a group of genes of genomic and mitochondrial genes was established as determinants of hearing loss. Another group of genes was associated with inner ear (vestibulum, labyrinth, endolymph) alterations followed by dizziness and tinnitus. Altogether, many studies suggest a multigenic interaction to predispose to develop Meniere’s disease.

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.

Low 5-year survival rate in laryngeal squamous cell carcinoma (LSCC) is to large extent attributable to high rate of recurrences and metastases. Despite the importance of the latter process, its complex genetic background remains not fully understood. Recently, we identified two metastasis-related candidate genes, DIAPH2 and DIAPH3 to be frequently targeted by hemizygous/homozygous deletions, respectively, in LSCC cell lines. They physiologically regulate such processes as cell movement and adhesion, hence we found it as a rationale, to study if tumor LSCC specimens harbor mutations of these genes and whether the mutations are associated with metastasizing tumors. As a proof of concept, we sequenced both genes in five LSCC cell lines derived from lymph node metastases assuming there the highest probability of finding alterations. Indeed, we identified one hemizygous deletion (c.3116_3240del125) in DIAPH2 targeting the FH2 domain. Moreover, we analyzed 95 LSCC tumors (53 N0 and 42 N+) using the Illumina platform and identified three heterozygous single nucleotide variants in DIAPH2 targeting conserved domains exclusively in N+ tumors. By combining these results with cBioPortal data we showed significant enrichment of DIAPH2 mutations (P = 0.036) in N+ tumors. To demonstrate the consequences of DIAPH2 inactivation, CRISPR/Cas9 editing was used to obtain a heterozygous DIAPH2+/- mutant HEK-293T cell line. Importantly, the edited line shows a shift from 'proliferation' to 'migration' phenotype typically observed in metastasizing cells. In conclusion, we report that DIAPH2 alterations are present primarily in metastasizing specimens of LSCC and suggest that they may contribute to the metastatic potential of the tumor.

In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-4991-4) contains supplementary material, which is available to authorized users.

Item does not contain fulltext Personalised medicine tumour boards, which leverage genomic data to improve clinical management, are becoming standard for the treatment of many cancers. This paper is designed as a primer to assist clinicians treating head and neck squamous cell carcinoma (HNSCC) patients with an understanding of the discovery and functional impact of recurrent genetic lesions that are likely to influence the management of this disease in the near future. This manuscript integrates genetic data from publicly available array comparative genome hybridization (aCGH) and next-generation sequencing genetics databases to identify the most common molecular alterations in HNSCC. The importance of these genetic discoveries is reviewed and how they may be incorporated into clinical care decisions is discussed. Considerations for the role of genetic stratification in the clinical management of head and neck cancer are maturing rapidly and can be improved by integrating data sets. This article is meant to summarise the discoveries made using multiple genomic platforms so that the head and neck cancer care provider can apply these discoveries to improve clinical care.

In recent years there are observed substantial changes in epidemiology of head and neck cancer. An incidence of laryngeal cancer is declining as a consequence of decreased tobacco smoking. Contrary, oropharyngeal cancer associated with HPV infection transmitted on sexual way is becoming much more frequent. The latter one is characterized with a better prognosis that most likely does not require intensive therapy. De-escalation of therapy in case of HPV-associated tumor is a matter of current studies.

We have turned our attention to CEACAM6 gene, already described as deregulated in various types of cancer. By using the expression microarrays performed on the set of 16 laryngeal squamous cell carcinoma (LSCC) samples: 11 cell lines and 5 primary tumors we have shown downregulation of CEACAM6 gene as compared to non cancer controls from head and neck region. CEACAM6 gene downregulation, further confirmed by quantitative PCR on 25 LSCC cell lines, was observed in cell lines derived from recurrent tumors in comparison to controls. A significant gene downregulation was observed in cell lines derived from advanced, high grade tumors in comparison to controls. Intrigued by the recurrent transcriptional loss of CEACAM6 we searched for the mechanism potentially responsible for its downregulation and hence we analyzed DNA copy number changes (a-CGH), promoter DNA methylation status and occurrence of gene mutations (in silico). Neither the analysis of gene copy number, nor the mutation screen has shown recurrent deletions or mutations, that could contribute to the observed downregulation of the gene. However, by using bisulfite pyrosequencing, we have shown DNA hypermethylation (mean DNA methylation > 78%) of CEACAM6 promoter region in 9/25 (36%) LSCC cell lines. Importantly, the 5-aza-2-deoxycytidine-induced inhibition of DNA methylation resulted in restoration of CEACAM6 expression in the two LSCC cell lines on mRNA level. In summary, we have shown that recurrent downregulation of CEACAM6 in LSCC is dependent on the gene’s promoter DNA methylation and is observed predominantly in large, poorly differentiated tumors and recurrences.

The treatment paradigms for head and neck squamous cell cancer (HNSCC) are changing due to the emergence of human papillomavirus-associated tumors (HPV-related), possessing distinct molecular profiles and responses to therapy. Retrospective studies have suggested that HPV-related HNSCCs are more frequently cured than those caused by tobacco. Current clinical trials focus on the reduction of treatment-related toxicity and the development of HPV-targeted therapies. New treatment strategies include: 1) dose reduction of radiotherapy, 2) the use of cetuximab instead of cisplatin for chemo-radiation 3) less invasive surgical options, i.e. trans-oral robotic surgery and trans-oral laser microlaryngoscopy, and 4) more specific treatment attempts, including immunotherapeutic strategies, thanks to increasing comprehension of the molecular background of HPV-related HNSCC. Whereas recently published data shed light on immune mechanisms, other studies have focused on specific vaccination against HPV-related HNSCC. A crucial problem is patient selection to the chosen bias. Truly HPV-related cancers (p16-positive and HPV DNA-positive) with biomarkers for good response to therapy could be included in randomized trials aiming for less severe and better tailored therapy.

Protocadherins are cell-cell adhesion molecules encoded by a large family of genes. Recent reports demonstrate recurrent silencing of protocadherin genes in tumors and provide strong arguments for their tumor supresor functionality. Loss of protocadherins may contribute to cancer development not only by altering cell-cell adhesion, that is a hallmark of cancer, but also by enhancing proliferation and epithelial mesenchymal transition of cells via deregulation of the WNT signaling pathway. In this study we have further corroborated our previous findings on the involvement of PCDH17 in laryngeal squamous cell carcinoma (LSCC). We used bisulfite pyrosequencing to analyze a cohort of primary LSCC tumors for alterations in PCDH17 promoter DNA methylation as an alternative gene inactivation mechanism to the homozygous deletions reported earlier. Moreover, we analyzed primary LSCC samples by immunohistochemistry for PCDH17 protein loss. We identified recurrent elevation of PCDH17 promoter DNA methylation in 32/81 (40%) primary tumors (P

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.

Despite that Robertsonian translocations (ROBs) are the most common chromosomal rearrangements in humans (1/1000 individuals), an exact breakpoint and the molecular mechanisms leading to their formation are still not well known. This is partly due to the fact that Human Genome Project did not provide any map or sequence for the acrocentric short arms. The main aim of our studies was to narrow the breakpoints in de novo arising and in familial cases of the most frequently occurring ROBs, using eight, previously not tested clones derived from 21p. Our results from PCR and FISH analysis showed that only the clones CR382285, CR382287, and a small fragment of CR382332 are retained in the examined ROBs. Moreover, interphase FISH on monochromosomal hybrids verified the orientation of studied clones in relation to centromeres of chromosomes 14 and 21. Given our results, we propose localization of the breakpoints in or nearby to clone CR382332. Summarizing, our results allowed to narrow the region where the breakpoints are localized and demonstrated that their position could be the same in all common ROBs. Electronic supplementary material The online version of this article (doi:10.1007/s10577-014-9439-3) contains supplementary material, which is available to authorized users.

Patient Female, 6 FINAL DIAGNOSIS: Phenotype-genotype discordance in congenital malformations with communication disorders resembling trisomy 18 (Edwards syndrome) Symptoms: - Medication - Clinical Procedure: - Specialty: Otolaryngology. Objective Congenital defects. Background Communication process disorders are very frequent in rare cases of chromosomal aberrations (deletions, insertions, and trisomies) such as Down syndrome (trisomy 21), Turner syndrome, Edwards syndrome (trisomy 18), or Patau syndrome (trisomy 13). Sometimes phenotype may delusively correspond to the characteristic features of a given syndrome, but genotype tests do not confirm its presence. Case report We present the case of a 6-year-old girl admitted to the Clinic of Phoniatrics and Audiology for the assessment of communication in the course of congenital malformations with phenotype characteristic for trisomy 18 (Edwards syndrome). Immediately upon birth, dysmorphic changes suggesting trisomy 18 (Edwards syndrome) were observed, but trisomy 18 was excluded after karyotype test results were normal (46, XX). Conclusions DISTURBED ARTICULATION WAS DIAGNOSED: deformed linguo-dental and palatal sounds, interdental realization with flat tongue of the /s/, /z/, /c/, /dz/, /ś/, /ź/, /c/, /dz/ sounds (sigmatismus interdentalis). Hearing loss was confirmed.

The role of human papilloma viruses (HPV) in malignant and nonmalignant ENT diseases and the corresponding epidemiological burden has been widely described. International head and neck oncology community discussed growing evidence that oral HPV infection contributes to the risk of oro-pharyngeal carcinoma (OPC) and recommended HPV testing as a part of the work up for patients with OPC. Polish Society of ENT Head Neck Surgery and National Chamber of Laboratory Diagnosticians have worked together to define the minimum requirements for assigning a diagnosis of HPV-related conditions and testing strategy that include HPV specific tests in our country. This paper briefly frames the literature information concerning low risk (LR) and high risk (HR) HPV, reviews the epidemiology, general guidance on the most appropriate biomarkers for clinical assessment of HPV. The definition of HPV-related cancer was presented. The article is aiming to highlight some of major issues for the clinician dealing with patients with HPV-related morbidities and to introduce the diagnostic algorithm in Poland.

Contains fulltext : 172579.pdf (Publisher’s version ) (Closed access) The knowledge of the biology of head and neck squamous cell carcinoma (HNSCC) has had relatively little impact on the improvement in oncologic outcome up to date. However, the identification of oncogenes and tumor suppressor genes (TSGs) involved in cancer progression contributes to the understanding of the molecular pathways involved in oncogenesis and could contribute to individual risk assessment and provide tools for improvement of treatment and targets for therapy based on the alterations in these pathways. The aim of this article is to review the chromosomal aberrations commonly found in HNSCC, to identify the genes in these chromosomal regions suggested to act as (candidate) oncogenes or TSGs, and to discuss the molecular mechanisms modulating their expression.

Squamous cell carcinoma of the head and neck (HNSCC) most frequently arise in the epithelial tissues of the upper aerodigestive tract. Patients with HNSCC, aged

Introduction Cigarette smoke and alcohol can generate reactive oxygen species, which may induce DNA double-strand breaks (DSBs), the most serious DNA lesion. In humans, DSBs are repaired mainly by non-homologous end joining and homologous recombination repair (HRR). Several polymorphisms in the DNA repair gene have been extensively studied in the association with various human cancers. In the present work we investigated the association between polymorphisms of two HRR genes, XRCC2 and RAD51, and tobacco- and alcohol-related larynx cancer in a Polish population. Material and methods Two polymorphisms of the XRCC2 gene, –41657C > T (rs718282) and 31479G > A (rs3218536), as well as one polymorphism of the RAD51 gene, –135G > C (rs1801320), were investigated by PCR-RFLP in 253 patients with larynx cancer and 253 age- and sex-matched non-cancer controls. Results Analysis of the gene-smoking and -drinking interactions revealed a weak association between larynx cancer and the –41657C > T polymorphisms of the XRCC2 gene among the moderate alcohol drinkers. The C allele of the –135G > C polymorphism of RAD51 increased cancer risk in the smoker group. Increased risk was also found for heavy drinkers. Additionally, there were no significant differences between distributions of genotypes in subgroups assigned to different TNM stages and grades. Conclusions The results indicated that the –135G > C polymorphism of the RAD51 gene may be associated with smoking- and drinking-related larynx cancer in Poland.

Although down-regulation of GNG7 in cancer was reported before, its role in carcinogenesis is poorly understood. It belongs to a family of large G-proteins that may be involved in cell-contact-induced growth arrest and function in tumor suppression. In the present study, we stained immunohistochemically 188 tumors derived from larynx or floor of the mouth for GNG7 protein and confronted it with clinicopathologic data. Moreover, we performed bisulfite pyrosequencing to analyze GNG7 promoter methylation. We identified recurrent loss of GNG7 protein expression in 68/188 (36%) cases and promoter hypermethylation in (42/98; 43%) primary tumors, predominantly in young patients (p

Thirteen laryngeal squamous cell carcinoma cell lines were recently studied by array comparative genomic hybridization (array-CGH) in order to identify recurrent DNA copy number alterations in the tumor genome. A highly amplified region 22q11.2 was found in two of the thirteen cell lines. Two established oncogenes CRKL and MAPK1 are localized in this region, but only CRKL was amplified in both cell lines. Therefore, to check if amplification of either CRKL or MAPK1 genes may be important in the pathogenesis of laryngeal squamous cell carcinoma, the DNA copy number and mRNA expression were measured in a cohort of 17 LSCC cell lines by quantitative real-time PCR (qPCR). For the CRKL gene gains of the copy number were found in 3/17 cell lines, while overexpression was found in 6/17 cell lines. Gains in the copy number for the MAPK1 gene were found in 1/17 cell lines, but overexpression was not detected in any cell line. A highly significant correlation between DNA copy number and expression for CRKL gene, but not for MAPK1 gene was established using the Pearson test. Thereafter, 46 primary samples of laryngeal cancer were tested by qPCR to check for possible gains in copy number of the CRKL gene. Gains were found in 3/46 cases. These results suggest that CRKL, but not MAPK1 is the target oncogene of the rare but recurrent amplification at 22q11.2 in laryngeal squamous cell carcinoma.

Many classical tumor suppressor genes (TSG) were identified by delineation of bi-allelic losses called homozygous deletions. To identify systematically homozygous deletions in laryngeal squamous cell carcinoma (LSCC) and to unravel novel putative tumor suppressor genes, we screened 10 LSCC cell lines using high resolution array comparative genomic hybridization (arrayCGH) and array based expression analysis. ArrayCGH identified altogether 113 regions harboring protein coding genes that showed strong reduction in copy number indicating a potential homozygous deletion. Out of the 113 candidate regions, 22 novel homozygous deletions that affected the coding sequences of 15 genes were confirmed by multiplexPCR. Three genes were homozygously lost in two cell lines: PCDH17/PCH68, PRR20, and PTPRD. For the 15 homozygously deleted genes, four showed statistically significant downregulation of expression in LSCC cell lines as compared with normal human laryngeal controls. These were ATG7 (1/10 cell line), ZMYND11 (BS69) (1/10 cell line), PCDH17/PCH68 (9/10 cell lines), and PTPRD (7/10 cell lines). Quantitative real-time PCR was used to confirm the downregulation of the candidate genes in 10 expression array-studied cell lines and an additional cohort of cell lines; statistical significant downregulation of PCDH17/PCH68 and PTPRD was observed. In line with this also Western blot analyses demonstrated a complete absence of the PCDH17 and PTPRD proteins. Thus, expression profiling confirmed recurrent alterations of two genes identified primarily by delineation of homozygous deletions. These were PCDH17/PCH68, the protocadherin gene, and the STAT3 inhibiting receptor protein tyrosine phosphatase gene PTPRD. These genes are good candidates for novel TSG in LSCC. (C) 2010 Wiley-Liss, Inc.

Introduction Laryngeal cancer (LSCC) is one of the most common tumours among head and neck squamous cell carcinomas. Recently, analysis of the meaning of epigenetic alterations in LSCC development has gained momentum. In this field we have shown recurrent overexpression of the miR-1290 in LSCC. Therefore, in this study we aim at clarifying the oncomiR functionality of miR-1290 by analysing its interaction with the transcriptional modulator MAF – a putative suppressor gene. Material and methods The identification of miR-1290 overexpression in LSCC was based on global miRNA expression profiles (Agilent) of 16 LSCC cell lines and 3 non-tumour controls which were validated on 50 tumour samples by real-time qPCR (RT-qPCR) with LNA modified primers. Target genes of miR-1290 were selected by in silico analysis using miRDB (TS >90) and miRWALK (gene listed in at least 2 in 4 databases). The influence of miR-1290 on its putative target gene MAF was analysed by transient transfection of 2 LSCC cell lines using miRNA inhibitor and successive expression analysis of MAF in the transfected cells by RT-qPCR and Western blot. To confirm the downregulation of MAF in primary LSCC we conducted RT-qPCR on 22 tumour samples and 5 non-tumour controls. In addition, the nuclear MAF protein abundance was evaluated by immunohistochemistry on tissue microarray containing 128 LSCC tumour samples and 19 tumour free resection margins. Results and discussions Based on the in sillico analysis MAF has been selected as a the putative miR-1290 target gene with high suppressor potential. Importantly, MAF is a transcription factor, involved in regulation of apoptosis and TP53 expression, processes deregulated in LSCC. Functional analyses, showed that inhibition of miR-1290 resulted in 3.47 fold increase in MAF mRNA expression and 50% increase of MAF protein abundance. Moreover, by using RT-qPCR we have shown recurrent MAF downregulation in LSCC tumour samples compared to non-tumour controls. In line with these findings we have indicated by immunohistochemical staining that 74/128 LSCC tumour samples were characterised by lack of nuclear expression of MAF in opposite to nuclear expression observed in 19/19 margins. Conclusion Our results demonstrate MAF as a new potential tumour suppressor gene epigenetically downregulated in LSCC by the overexpression of miR-1290. Further studies are required for fully understanding the role of MAF in LSCC pathogenesis.

The aim of the study was to assess genotoxicity of a chromium(III) propionate complex in rat's peripheral blood lymphocytes by the comet assay. The study was carried out on 18 12-weeks old female Wistar rats that were divided into three equal groups (six animals each): control (0), control-Cr(VI) and Cr(III)-tested rat fed ad libitum a basal diet and the diet supplemented either with 10 mg Cr(VI)/kg diet (given as K(2)Cr(2)O(7), equivalent of 1mg/kg body mass/day) or 1000 mg Cr(III)/kg diet (given as [Cr(3)O(O(2)CCH(2)CH(3))6(H(2)O)(3)]NO(3)), equivalent of 100mg Cr/kg body mass/day) for 4 weeks. High doses of supplementary Cr(III) were found to not affect body mass gain, feeding efficiency ratio and internal organ masses. Treatment of rats with the Cr(III) propionate complex, in contrast to Cr(VI), did not affect significantly the comet assay results in lymphocytes, which suggests that the compound does not exert genotoxic effects in rats.

CDKN2A gene belongs to the genes involved in cell cycle regulation. When is absent or inactivated by mutation or promoter hypermethylation a cell may undertake an uncontrolled proliferation. Inactivation of CDKN2A gene is observed in many human malignancies, including larynx cancer. In this study we investigated mutations in exon 1 and exon 2 of CDKN2A gene in a large group of 390 laryngeal cancers. We found 40 different alterations (17%) and nearly half of them was not described previously. Out of these alterations two transversions in codon 108: c.322G>C (Asp108His) and c.322G>T (Asp108Tyr) as well as a G>A transition in codon 110 (Trp110X) were found more frequently (altogether: 7 cases in codon 108 and 10 cases in codon 110). This result, concerning the location of these codons in the ankyrin repeat structures, may suggest that these two codons may be critical hot-spots in larynx carcinogenesis.

Carcinogenesis may result from abnormal methylation of cancer-related genes regulatory sequence. Though, the polymorphic variants of genes encoding enzymes of folate and methionine metabolism may have an effect on DNA methylation. Using PCR-RFLPs, we examined the polymorphism distribution of genes encoding methionine synthase (MTR); 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase (MTHFD1); and methylenetetrahydrofolate reductase (MTHFR) in patients with larynx cancer (n = 131) and controls (n = 250). Patients with MTR 2756AG or GG genotypes displayed a 1.856 -fold increased risk of larynx cancer (95% CI = 1.1860–2.903, P = 0.0076). However, we did not observe an increased risk for the homozygous GG genotype OR = 1.960 (95% CI = 0.6722–5.713, P = 0.2535). Moreover, we did not observe statistical differences in distribution of MTHFR 677C>T, 1298A>C and MTHFD1 1958G>A allele and genotype frequencies in patients and controls. Our findings confirm the significance of the role of the methyl cycle in etiopathogenesis of laryngeal cancer.

Summary The amino acid substitution Arg72Pro in the TP53 protein has an impact on the biochemical and biological activity of this protein, and is associated with several types of cancers. However, the Arg72Pro polymorphism exhibits inconsistent contribution as a risk factor in various cancer types. Therefore, using PCR-RFLPs, we investigated the distribution of Arg72Pro genotypes and alleles in patients with laryngeal cancer ( n = 123) and controls ( n = 300) in Poland. We observed that patients with the Pro/Pro and Arg/Pro TP53 genotypes displayed a 1.755-fold increased risk of laryngeal cancer (95% CI = 1.149–2.680, P = 0.0099). However, we did not find a significant increase in laryngeal cancer risk for the homozygous Pro/Pro TP53 genotype OR = 2.093 (95% CI = 1.046–4.192, P = 0.0530). This result suggests that the TP53Pro variant may contribute to the risk of laryngeal cancer development in Polish patients.

Mutations in mitochondrial DNA are associated potentially with nonsyndromic and aminoglycoside-induced hearing loss. Several nucleotide changes associated with hearing impairment were described; however, a variable frequency of deafness-associated mutations in different populations has been observed. The aim of the present study was to determine the frequency of pathological mutations in mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes in a group of 500 individuals representative of the general population of Poland. Mutational screening of 12S rRNA revealed the presence of three deafness-associated mutations, A827G, T961C, and A1555G, and one potentially pathogenic substitution, T669C. The carrier frequency of pathological mutations was estimated to be 1.2% (6/500) in the general Polish population. A deafness-associated G7444A mutation in the precursor of tRNA(Ser(UCN)) gene was identified in 8/500 (1.6%) unrelated blood donors. Seven nucleotide changes identified in 12S rRNA (G709A, G750A, G930A, T1243C, T1420C, and G1438A) and tRNA(Ser(UCN)) (C7476T), based on a frequency exceeding 1.0%, were considered as polymorphisms of 12S rRNA and tRNA(Ser(UCN)) in the studied population. Mitochondrial 12S rRNA gene seems to be the hot spot for deafness-associated mutations in the Polish population. The relatively high carrier frequency of tRNA(Ser(UCN)) G7444A (1/62) suggests that this substitution might be a nonpathogenic polymorphism in the Polish population.

Tobacco smoke-related products and ethanol would induce oxidative modifications to the DNA bases, thereby contributing to larynx cancer. Human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) deals with oxidative DNA damage, and the base changes in the hOGG1 gene may alter the susceptibility of the human cells to tobacco smoke-related compounds and/or ethanol. In the present work, we investigated the association between smoking, drinking or the Ser326Cys polymorphism of the hOGG1 gene and the risk of larynx cancer in a Polish population. It has been reported that the Ser326 allele exhibits higher activity than the Cys326 variant. In this study, 253 age-matched controls and 253 patients with larynx cancer were enrolled. The polymorphism was determined with DNA from blood lymphocytes by polymerase chain reaction. The frequencies (%) of the genotypes were Ser/Ser 65.6, Ser/Cys 30.4, and Cys/Cys 4.0 in the controls and those in patients were 55.7, 36.0 and 8.3, respectively. Stratification of individuals according to their smoking and drinking habits indicated that these habits might be significant risk factors in larynx cancer. The Ser/Cys and Cys/Cys genotypes are significantly associated with the increased risk of larynx cancer. These genotypes increased the risk ratio of larynx cancer among heavy smokers, but did not change the risk in former smokers and moderate smokers. These genotypes also increased the risk of larynx cancer in moderate and heavy drinkers. Therefore, the Cys326 allele of the hOGG1 gene may increase the risk of larynx cancer associated with smoking or alcohol consumption.

The introduction of prognostic tools to evaluate rehabilitation progress in cochlear implant patients (CI patients) is of great importance. The authors attempted to verify whether the identified 35delG mutation in the GJB2 gene can serve as a valuable indicator for rehabilitation progress of CI patients. A group of 51 subjects was studied. Molecular analysis was based on the identification of 35delG in GJB2. Logopedic assessment was performed with a non-verbal test of seven sounds, evaluating detection, discrimination and identification of the sounds during the first, third and sixth months after implantation. Results indicated that patients with GJB2-related deafness (DFNB1)s achieve better results in rehabilitation, but only at the early stages of rehabilitation. Prolonged rehabilitation equalised differences, which, subsequently, excluded this marker as an indicator for rehabilitation evaluation. Copyright © 2008 John Wiley & Sons, Ltd.

Introduction Hereditary haemorrhagic telangiectasia (HHT) known also as Rendu-Osler-Weber syndrome is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in ENG (endoglin, 9q34.1) or ALK-1 gene (the activin receptor-like kinase 1, 12q13). ENG and ALK-1 are found associated with two disease subtypes designated as HHT1 and HHT2, respectively. Subtype HHT1 remains in the frame of interest of laryngology because of frequent bleeding in head and neck region. Material and method The study was designed to identify a genetic background in a large family (29 individuals) with diagnosed HHT. Pedigree analysis showed autosomal dominant pattern of inheritance. Study design comprised segregation analysis to determine locus with subsequent direct sequencing of the gene. Four microsatelite markers (d9s61, d9s65, d12s368, d12s347) with high frequency of heterozygosity in population study were used. Results The results concerning heterozygosity ranged from 15% to 53%. The established differences were not sufficient enough to indicate co-segregation of the studied loci. DNA sequence analysis in exon 11 of ENG gene did not reveal mutations. The latter result could be explained by an occurrence of mutations in other exons of ENG. Conclusions The study requires continuation for gene identification and precise genotype-phenotype correlation aiming for an improvement of HHT1 therapy.

Patients with squamous cell carcinoma of the head and neck (HNSCC) aged less than 45 years are categorized as young adults (YA) and in opinion of many authors in comparison to older (typical) patients (OP) are characterized with more serious form of the disease and often lack the classical risk factors associated with the illness. Hence, there is a need of an exact clinical analysis and a search for additional causative factors. The purpose of this study was to estimate the role of genetic factors in the etiology of HNSCC in YA. Studies carried out on 60 patients of the study group (YA) and 72 older control patients were directed to: (1) a degree of spontaneous and induced chromosome breaks estimated by bleomycin test, (2) a degree of spontaneous and induced DNA damage and a potential of DNA repair determined by comet assay and (3) polymorphism of genes of glutathione transferases M1 and T1, responsible for detoxification of metabolites of carcinogens of tobacco smoke, studied by PCR-based genotyping. The level of chromosome breaks (spontaneous and induced), the level of DNA damage (spontaneous and induced), DNA repair potential and the distribution of polymorphic variants of GSTT1 gene are not significantly different in YA and in OP, which suggests that these factors do not appear the causative factors for HNSCC in young age. The significant risk factor of HNSCC in YA may be GSTM1 null genotype, which may cause the defective detoxification of metabolites of polycyclic aromatic hydrocarbons of tobacco smoke.

UNLABELLED Squamous cell carcinoma of the head and neck (HNSCC) is the malignant tumor arising most frequently in non-keratinized epithelial tissue of the upper part of the respiratory or gastrointestinal tracts. These tumors develop most commonly in the sixth or seventh decade of life and significantly less frequently in patients younger than 45 years defined as young adults. In literature, there are still many controversies concerning the origin of these tumors in this group of patients. AIM To estimate the role of exogenous and epidemiological factors in etiology of HNSCC in young adults. MATERIAL AND METHODS The study group consisted of 95 young adults ( 45 years) with HNSCC, who were treated in the Department of Otolaryngology and Laryngological Oncology of Karol Marcinkowski University of Medical Sciences in Poznan during the period 2000-2004. The analysis was based on etiological questionnaires and the medical records of patients. RESULTS AND CONCLUSIONS 1. A significant role of typical exogenous factors (active and passive exposure to tobacco smoke and alcohol abuse) was established in the etiology of HNSCC in young adults. 2. Daily active exposure to tobacco smoke in young adults is nearly the same as in older patients, but daily passive exposure is higher than in older patients. 3. Significantly higher alcohol consumption in young adults than in older patients, found in this study, may be one of the reasons of HNSCC onset in young age. 4. Occupational exposure to the HNSCC causative factors in young adults is not higher than in older patients. 5. A frequency of familial cancer in young adults with HNSCC seems to be higher than in older patients, but further observations are needed.

Summary Squamous cell carcinoma of the head and neck (HNSCC) is the malignant tumor arising most frequently in non-keratinized epithelial tissue of the upper part of the respiratory or gastrointestinal tracts. These tumors develop most commonly in the sixth or seventh decade of life and significantly less frequently in patients younger than 45 years defined as young adults. In literature, there are still many controversies concerning the clinical course of these tumors in this group of patients. Aim Clinical and histopathological analysis of HNSCC in young adults. Material and methods The study group consisted of 95 young adults (≤45 years) with HNSCC and the control group of 95 older patients (>45 years) with HNSCC, who were treated in the Department of Otolaryngology and Laryngological Oncology of Karol Marcinkowski University of Medical Sciences in Poznan during the period 2000–2004. The analysis was based on medical records of patients. Results and conclusions 1. In the young adults group and particularly in patients younger than 40 years there is a higher percentage of patients with oral cancer and lower percentage of patients with larynx cancer in comparison to older patients group, but in all analysed groups larynx cancer is the most frequent localization. 2. The tumors in young adults are clinically more advanced than in older patients, because young adults tend to delay the visit to physician, in spite of evident clinical symptoms. 3. HNSCC in young adults are histological more mature and less malignant.

Head and neck cancers are one of the most frequent cancers worldwide. This paper attempts to evaluate disturbances of homeostasis of the necessary elements (calcium, magnesium, zinc, copper, iron, manganese) and changes in the levels of toxic metals (lead, cadmium, cobalt, chromium VI) in hair of patients with head and neck cancers, as well as people without a diagnosed neoplastic disease. In order to quantify the necessary elements and toxic metals, a method using ICP-MS and ICP-OES techniques had been developed and validated. The studies have shown that patients with head and neck cancer used to drink alcohol and smoked much more frequently than healthy individuals, both in the past and presently. Statistically significant differences in concentrations of average metal content in the group of patients with head and neck cancers compared to the control group were confirmed. Significant differences in metal content between the group of patients with head and neck cancers and healthy individuals were found which enabled distinguishing between the study groups. To this end, a more advanced statistical tool, i.e. chemometrics, was used. The conducted research analyses and the use of advanced statistical techniques confirm the benefits of using alternative material to distinguish the patients with head and neck cancers from the healthy individuals.

Trace elements have an impact on numerous physiological processes. The monitoring of their levels in the organism allows you to detect not only their deficiencies, but also several illnesses. The aim of this study was to compare the levels of essential elements (calcium, magnesium, zinc, copper, iron, manganese) in hair, nails and serum of both patients with laryngeal cancer and healthy people. The determination of six metals was performed by an inductively coupled plasma mass spectrometry (ICP-MS) and an inductively coupled plasma optical emission spectrometry (ICP-OES). The concentration of essential elements in hair and nails of the control group was statistically significantly higher than in the group of patients with laryngeal cancer. In the case of serum, differences were found between the patients and controls in respect of the level of three metals. The results of principal component analysis (PCA) revealed the strong and similar clustering behavior of essential elements in hair and nails. The metals did not correlate between two alternative materials. The present study indicated that, using the level of essential elements in hair and nails as a basis, it is possible to distinguish cancer patients from healthy people. The alternative materials are independent of homeostasis and therefore seem to be more useful in the detection of diseases and mineral deficiencies in human than the classical biological materials, such as blood.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. AhR together with ARNT, AhRR, HIF1alpha represent a novel basic helix-loop-helix/PAS family of transcriptional regulators. Their interplay may affect the xenobiotic response. In this study, the effect of i.p. administration of different AhR ligands on the expression of AhR, AhRR, ARNT, HIF1alpha and CYP1A1 and NAD(P)H: quinone oxidoreductase (NQO1), the enzymes controlled by AhR were examined in Sprague-Dawley rat liver. Quantitative real-time RT-PCR analysis revealed no changes in the mRNA expression of ARNT and HIF1alpha following 3-methylcholanthrene (3-MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or beta-naphthoflavone (BNF) treatment. AhRR expression was affected by TCDD but not by BNF and 3-MC. Expression of AhR mRNA and of the markers of its activation, CYP1A1 and NQO1, was significantly increased by administration of TCDD, 3-MC and, to lower extent, BNF. These results indicate that binding of the ligands to AhR up-regulates the mRNA transcription not only of CYP1A1 and NQO1, but also of AhR itself. The level of AhR induction depends on the potency of xenobiotic metabolizing enzymes inducer.