DIAPH2 alterations increase cellular motility and may contribute to the metastatic potential of laryngeal squamous cell carcinoma.

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.

Recurrent CDK1 overexpression in laryngeal squamous cell carcinoma.

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.

Moving towards personalised therapy in head and neck squamous cell carcinoma through analysis of next generation sequencing data.

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.

Polymorphisms of DNA repair genes and risk of squamous cell carcinoma of the head and neck in young adults.

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 <45 years are categorized as young adults (YA). They are characterized by more severe form of this disease and often lack of classical, causative risk factors (tobacco smoking, alcohol abusing) in comparison to older (typical) patients (OP). The study purpose was to establish an anticipated protective role of DNA repair genes polymorphisms against cancer-causing agents. It was assumed that the polymorphisms in these genes may have a significant role in the etiology of HNSCC in YA. Studies were carried out on three groups: YA group with HNSCC (n = 90), young healthy group without cancer (YH, n = 160) and OP with HNSCC (n = 205). Three polymorphisms in DNA repair genes were analyzed: XPD ex23: A35931C, XRCC1 ex10: G28152A, and XRCC3 ex7: C18067T. The choice of these genes was connected with their involvement in three different DNA repair pathways. Genotyping was carried out by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) technique. Statistical analysis included: calculation of odds ratio (ORs), 95 % confidence intervals (CIs) and p value. There was no significant difference in the distribution of XPD genotypes in YA compared to OP or YH. The XRCC1 AA genotype variant was observed less frequently in HNSCC YA (4.7 %) than in YH and in OP group (17.1 and 10.8 %, respectively). XRCC3 CT genotype variant was observed more frequently in HNSCC YA (61.8 %) than in YH (36.3 %) and this result is statistically significant. This variant was associated with the borderline increased risk of HNSCC development in an early age, however, a similar tendency was not observed in case of double mutated TT variant. The established differences of genotypes distribution do not seem to differentiate substantially YA and OP in head and neck cancer risk.

Polymorphisms of the DNA repair genes XRCC1 and ERCC4 are not associated with smoking- and drinking-dependent larynx cancer in a Polish population.

BACKGROUND: Tobacco smoking and alcohol drinking generate oxidative DNA damage and may contribute to larynx carcinogenesis. The X-ray repair cross complementing 1 (XRCC1) and excision repair cross-complementing rodent repair deficiency, complementation group 4 (ERCC4(XPF)) genes are important components of DNA excision repair systems, which repair DNA damage induced by various factors, including tobacco smoking and alcohol. AIM: To investigate the association between the genotypes of the XRCC1-Arg399Gln (rs25487) and ERCC4-Arg415Gln (rs1800067) polymorphisms and smoking- and drinking-related larynx cancer in a Polish population. METHODS: The polymorphisms were determined by PCR-RFLP method in 253 patients with squamous cell carcinoma of the larynx and 253 sex- and age-matched controls. RESULTS: We did not find any association between the investigated polymorphisms and larynx carcinoma, dependent on either smoking or drinking status. No association was found between these polymorphisms and larynx cancer grade, stage or age at diagnosis. CONCLUSIONS: The results indicated that Arg399Gln polymorphism of XRCC1 gene and Arg415Gln polymorphism of ERCC4 gene may not be associated with smoking- and drinking-related larynx cancer in Polish population.

Nonrandom DNA copy number changes related to lymph node metastases in squamous cell carcinoma of the lung.

Lung cancer is one of the most common malignancies and cancer-related death worldwide. Lymph node metastasis is the main cause of treatment failure. Although many studies were performed to evaluate genetic events associated with development and progression of lung cancer, molecular mechanism still remains poorly defined. In the present study, using comparative genomic hybridization (CGH) technique, we described the pattern of DNA copy number changes in a cohort of 42 primary squamous cell carcinomas (SCC) of the lung. A direct comparison of nonmetastatic (TxN0M0) and metastatic (TxN1-2M0) tumors was performed to define chromosomal imbalances related to lymph node metastases. Some genetic alterations were observed more frequently in metastatic than in non-metastatic tumors, including losses at 11q, 16p, 16q, 19p and gains at 4q, 7q, 12p, 13q, 18p. The gain at 7q with the smallest common altered region 7q31.2-q32, was found to be directly associated with lymph node involvement (p=0.0407). We suggest that the established chromosomal region harbors two putative tumor suppressor genes WNT2 and c-Met. An overexpresion of these genes seems to be involved in inducing the invasive growth and metastatic potential of SCC of the lung.

Chromosomal gains and losses indicate oncogene and tumor suppressor gene candidates in salivary gland tumors.

The incidence of salivary gland tumor in Poland is growing in the last two decades. Simultaneously a progress in understanding the genetic mechanisms of formation of this tumor was achieved by detecting several genes like PLAG1 involved in its pathogenesis. In this study we perform a whole genome, CGH analysis with the aim to identify recurrent, chromosomal copy number changes possibly indicating novel tumor suppressor gene or oncogene loci. 29 salivary tumor samples: Cystadenolymphoma-warthin (15) and adenoma polymorphum (14) located in the parotid (27) and submandibular gland (2) were collected and CGH was performed. The established copy number profiles were compared in order to asses the smallest common region of gains and losses. The delineated regions were further analyzed with the UCSC Genome Browser on Human Mar. 2006 Assembly to asses their gene content. Altogether, salivary gland tumors presented a different aberration pattern than these reported for head and neck squamous cell carcinoma (HNSCC) but no significant differences were observed between Warthin and adenoma polymorphum tumors. Moreover, several potential tumor suppressor genes and oncogenes were identified in the smallest, common altered regions. We show a frequent deletion of the harakiri gene (12q24.2) in 12/29 tumors and TP53 gene (17p13.1) in 11/29 tumors as potential tumor suppressors in salivary gland cancers. Besides, we detected a frequent amplification of the 13q22.1-22.2 region in 13/29 cases harboring the KLF5 and KLF12 genes. KLF5 regulates the expression of survivin, an oncogene widely expressed in the majority of human cancers. The observed alterations may indicate important genetic events in the formation of salivary gland tumors. Especially the amplification in 13q may be a mechanism contributing to the expression of survivin and tumor progression.

Second primary tumors (SPT) of head and neck: distinguishing of "true" SPT from micrometastasis by LOH analysis of selected chromosome regions.

The reason of treatment failures in head and neck tumors is often connected with the appearance of second primary tumors (SPT). Three mechanisms of SPT development of clonal or non clonal secondary tumors were described: 1. via micrometastases (clonal); 2. from a common carcinogenic field - Second Field Tumors (SFT - partially clonal); 3. via independent events (from different carcinogenic fields - "true" SPT - not clonal). Assessing the clonality of diagnosed tumors carries important clinical implications including chemoprevention, radiotherapy and general patient management. In this study a set of 12 microsatellite markers was used to find similarities and/or differences in allelic imbalance patterns between 22 pairs of tumors (the first tumor designate as index and SPT). The aim of the study was to identify a potential clonal origin and progression within given pairs of tumors. The results indicate that within the tumors diagnosed by clinical examination as SPT at least two mechanisms mentioned above should be taken into account as 6/23 (26%) were clonally unrelated ("true" SPT) and 3/23 (13%) carried clonal genetic changes (formation by micrometastasis or SFT). In 14/23 (61%) cases the results were insufficient or ambiguous to determine the clonality status. The final results indicate the complexity of carcinogenesis in these tumors and thus stress that clinical diagnosis of second primary tumors should be considered carefully.

[Analysis of chromosome instability as a genetic risk factor of laryngeal cancer]. / Analiza niestabilnosci chromosomowej pod katem oceny ryzyka genetycznego wystapienia raka krtani.

The bleomycin test was established in the laboratory of Hsu et al. (M.D. Anderson Cancer Center, Houston, Texas) for quantitative estimation of hidden chromosome instability. The article presents a concept, method and applicability of the test. Chromosome sensitivity initially studied in respect to estimate a general genetic risk to develop cancer was found as a technique of an extended potential to study a genetic risk of familial cancer, young adults & neck cancer and other associations.

[An attempt to identify the most frequent genomic mutations responsible for isolated deafness in patients after cochlear implantation]. / Próba identyfikacji najczestszych mutacji genowych odpowiedzialnych za gluchote izolowana u pacjentów nieslyszacych zaopatrzonych w implant slimakowy.

The aim of this study was to identify subjects with 35delG mutation of GJB2 gene as the most frequent genetic cause of deafness. Deaf patients receiving cochlear implantation at the ENT Clinic at University of Medical Sciences in Poznan and their family members were recruited to the study. Peripheral blood lymphocytes DNA was amplified in allele-specific PCR and analysed for single strand conformation polymorphism (SSCP) to detect mutation at DFNB1 locus. 35delG mutation at both alleles was found at 42.9% of deaf patients and 29.4% of health relatives were found to be carrier of the mutation at one allele. The study is thought to be a first step in analysis of typical mutations in Polish deaf population.

[Paclitaxel (Taxol) in chemotherapy of head and neck cancer--just another proposal or breakthrough?]. / Paklitaksel (Taxol) w chemioterapii nowotworów glowy i szyi--kolejna propozycja czy przelom?

The examples of so far application of taxol in head and neck cancer are not completely clear. This is the first representative of a totally new group of cytostatics known as taxones. Presently, paclitaxel (TAXOL) is the anti-cancer drug commonly used in ovary cancer patients, resistant to platinum analogues. Taxones have the different functioning than the other anti-cancer drugs. They are responsible for originating microtubules and their further stabilisation. Moreover, paclitaxel (TAXOL) is a chemical compound sensitizing tumor cells for ionizing radiation. The authors are trying to specify the role of taxones nowadays and for the future use.

[Comparison of DNA adducts between oral, pharyngeal and larynx cancer]. / Porównanie zachowania sie poziomu adduktów kancerogen: DNA w rakach jamy ustnej, gardla i krtani.

The results concerning examination of DNA adducts in oral (23 patients), pharyngeal (23 patients) and larynx cancer (10 patients) subjects are presented. DNA adduct levels were compared in respect to anatomical structure (primary tumour location), number of cigarettes smoked, TNM stage, and age of patients. DNA was isolated from removed tissue (tumour and non-tumour surrounding tissue) using detergent/phenol extraction. 32P-postlabelling assay including nuclease P1-enhancement modification was applied. Aromatic DNA adducts were found in all studied tissues. Total DNA adduct levels (tumour and non-tumour tissues) was lowest in larynx cancer, higher in oral cancer and highest in pharyngeal cancer. There were no influence of age into formation of DNA adducts. The higher level of DNA adducts was found in tumour tissue of oral cancer in the group of smokers with metastasis into lymph nodes.

Association of arylamine N-acetyltransferase (NAT1 and NAT2) genotypes with urinary bladder cancer risk.

Arylamines are known bladder carcinogens deriving from tobacco smoke and environmental pollution. Arylamines are metabolised by NAT1 and NAT2 polymorphic enzymes in reactions of carcinogen activation and detoxification. We analysed genetic polymorphisms in both NAT1 and NAT2 genes in 56 bladder cancer patients and 320 healthy patients. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 (six alleles) and NAT2 (four alleles) by PCR-RFLP. A weak association between NAT1 and NAT2 genotypes and bladder cancer risk was found when the genotypes were estimated separately (odds ratio OR 1.2, 95%CI 0.7-2.0, and OR 1.3, 95%CI 0.7-1.9, respectively). Almost all NAT1 genotypes possessing at least one "risk" *10 allele were more frequent in the bladder cancer group than in the control group. There was also an increased frequency of "risk" genotypes along with increased cigarette smoking in bladder cancer patients. The coincidence of NAT1-fast/NAT2-slow appears as a potential risk factor for urinary bladder cancer (OR 1.5, 0.8-3.0), as compared with the other genotype combinations.

[Analysis of aromatic DNA adducts in oral cavity and pharyngeal cancer]. / Analiza aromatycznych adduktów kancerogen: DNA w rakach jamy ustnej i gardla.

An exposure to tobacco smoke carcinogens is followed by an interaction of chemical carcinogens with DNA molecule resulted by the formation of carcinogen: DNA adducts. The study subjects were 40 oral and pharyngeal cancer patients with primary tumours diagnosed as squamous cell carcinoma. The biological samples purchased for analysis included tumour biopsy, the surrounding tissue (histopathologically recognised as non-malignant) and peripheral blood leucocytes. For DNA adducts analysis first DNA was isolated using phenol detergent extraction followed by 32P-postlabelling assay including P1 nuclease enhancement. Aromatic DNA adducts were found in all studied tissues. The average levels of DNA adducts in tumour and non-tumour tissues were found higher than in leucocytes. Biopsies from pharynx contained higher levels of DNA adducts than from oral cavities. Confounding effect of tobacco smoking and alcohol consumption on DNA adducts level was observed but it has not reached statistical significance. Formation of DNA adducts was not depended on such factors as patients age, sex, pulmonary tuberculosis, cancer progression (TNM), environmental pollution and mechanical irritation.

Analysis of aromatic DNA adducts in laryngeal biopsies.

Epidemiological studies have confirmed the correlation between tobacco smoking, environmental pollution and the incidence of cancers of the respiratory tract. The occurrence of laryngeal cancer in Poland is relatively high compared to other European countries. Since 1969 the mortality related to larynx cancer appears to be increasing. Tobacco smoke contains an abundance of such carcinogenic compounds as polycyclic aromatic hydrocarbons (PAH), aromatic amines and N-nitrosoamines, which can react with DNA and form adducts. We analyzed aromatic DNA adducts in laryngeal tissues from patients with primary laryngeal, which was confirmed histopathologically to be squamous cell carcinoma. The group consisted of 33 patients (5 women and 28 men). Total laryngectomy was performed in patients. A detergentphenol method was used for DNA isolation. Aromatic DNA adducts were analyzed by a 32P-postlabelling technique with butanol extraction and high performance liquid chromatography. The presence of aromatic DNA adducts was demonstrated in all tissues. Large interindividual differences of DNA adduct levels were seen in each tissue studied. There was a higher mean level of DNA adducts in interarytenoid area non-tumors (51.96/10(8) +/- 91.71 NN) than in non-tumor tissue elsewhere (46.91/10(8) +/- 46.36 NN) and tumor tissue (43.52/10(8) +/- 45.88 NN). Adduct levels were correlated with age, sex, cigarette smoking and TNM stage.

[Usefulness of chromosome alteration analysis in cell lines derived from laryngeal tumors for evaluation of laryngeal neoplasms]. / Przydatnosc analizy uszkodzen chromosomów w liniach komórkowych wyprowadzonych z nowotworów krtani w ocenie przebiegu raka krtani.

A classical cytogenetic analysis was applied to analyse karyotypes of 9 cell lines derived from laryngeal cancer. The most frequent aberration was a deletion of the whole sex chromosome Y. An abundance of somatic chromosome alterations was further analysed to find correlation with tumour staging and grading. A conventional cytogenetic analysis seems to be not sufficient to recognize chromosome alterations specific for a given tumor stage. On the other hand, an analysis in respect to histologic grading has indicated for an association between rearrangement of 9 chromosome and a high tumor aggressiveness. It seems that a combination of conventional cytogenetics with molecular methods (FISH, CGH) would be helpful in diagnosing of laryngeal cancer.

Carcinogen: DNA adducts in tobacco smoke-associated cancer of the upper respiratory tract.

Mortality connected with tobacco smoke-associated laryngeal cancer in Poland markedly exceeds the relevant epidemiological data from other European countries. The main groups of genotoxic agents considered as potential carcinogens present in tobacco smoke are polycyclic aromatic hydrocarbons, aromatic amines, N-nitrosoamines and reactive oxygen species. Aromatic DNA adducts, N7-alkylated guanosines and oxidative DNA damage derived from tobacco smoke exposure were detected in laryngeal and oral (tumour and non-tumour) biopsies, and white blood cells of cancer subjects. Further, DNA lesions were analysed to estimate the significance of such confounders as intensity of smoking, subject's sex, age, topography of larynx, cancer staging and genetic factor. The number of cigarettes smoked per day was found to be the main determinant of an individual's DNA adduct level. The occurrence of DNA lesions was established as a reliable marker of former exposure to tobacco smoke genotoxicants. On the other hand, a comparison of DNA lesion levels in various regions of larynx indicates limited usefulness of DNA adduct analysis as an estimate of cancer risk. For a better risk estimation one has to take into account DNA lesions in proto-oncogenes and tumour suppressor genes and the efficacy of DNA repair. Altogether, DNA adducts formation and removal has to be considered as a single stage in the multistep carcinogenesis.

Molecular and cellular alterations in tobacco smoke-associated larynx cancer.

Tumours of head and neck belong to the most frequent types of cancer world-wide. In Poland, mortality from larynx cancer among males has been continuously increasing during the last decades up to 8.4 deaths per 100,000 men in 1993, which exceeds epidemiological records from other countries. The aetiology of laryngeal cancer is strongly associated with exposure to carcinogens present in tobacco smoke. The review describes a sequence of molecular and cellular events from carcinogenic exposure, DNA adduct formation, detection of mutations in the p53 gene, loss of heterozygosity (LOH) in chromosomal loci encoding the p53 and p16 genes, and loss of control of the cell cycle. The section concerning DNA adducts includes a discussion of the role of such confounders as exogenous exposure, the age and sex of the subject, and disease progression. The significance of genetic factors as individual risk determinants is discussed in relation to bleomycin-induced chromosome instability and in connection with the occurrence of defects in genes encoding detoxifying enzymes. The question concerning the substantial difference between men and women in larynx cancer morbidity and mortality remains open, even when the significantly higher adduct formation in male DNA compared with female material was taken into account. Preliminary experiments suggest a role of the frequently observed loss of the Y-chromosome.

In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues.

Malathion [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorodithioate] is a commonly used organophosphorus insecticide reported to be genotoxic both in vivo and in vitro, but the reports are conflicting. In order to elucidate the genotoxic potency of the main compounds present in commercial preparations of malathion, the DNA-damaging effect of this insecticide, its major metabolite malaoxon [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorothiolate] and its isomer isomalathion [S-(1,2-dicarboethoxyethyl)O,S-dimethyl phosphorodithioate], all at purity of at least 99.8%, was investigated by use of the alkaline single cell gel electrophoresis (comet assay). Freshly isolated human peripheral blood lymphocytes were incubated with 25, 75 and 200 microM of the chemicals for 1 h at 37 degrees C. The concentrations used are comparable to those found in blood following various non-lethal human exposures to pesticides. Malathion did not cause any significant changes in the comet length of the lymphocytes, throughout the range of concentrations tested. Malaoxon and isomalathion introduced damage to DNA in a dose-dependent manner. The effect induced by malaoxon was more pronounced than that caused by isomalathion. Treated cells were able to recover within a 60-min incubation in insecticide-free medium at 37 degrees C except the lymphocytes exposed to malaoxon at 200 microM, which did not show measurable DNA repair. The latter result suggests a considerable cytotoxic effect (cell death) of malaoxon at the highest concentration used. The reported genotoxicity of malathion might, therefore, be a consequence of its metabolic biotransformation to malaoxon or the presence of malaoxon and/or isomalathion as well as other unspecified impurities in commercial formulations of malathion. In this regard, the results of our study clearly indicate that malathion used as commercial product, i.e., containing malaoxon and isomalathion, can be considered as a genotoxic substance in vitro. This means that it may also produce DNA disturbances in vivo, such as DNA breakage at sites of oncogenes or tumor suppressor genes, thus playing a role in the induction of malignancies in individuals exposed to this agent. Therefore, malathion can be regarded as a potential mutagen/carcinogen and requires further investigation.