Miroslav Vukadinovic

Genomic instability and tumor-specific DNA alterations in oral leukoplakias

Leukoplakias, clinically identifiable premalignant lesions, often precede oral squamous cell carcinoma (OSCC). Identification of leukoplakias that have the potential for transformation to malignancy is a key clinical problem. The aim of this study was to assess genomic instability, and to detect tumor-specific genomic alterations, in leukoplakias. Genomic instability was analyzed by comparing the DNA fingerprints of 32 leukoplakias with those of paired normal tissue. In addition, the mutational status of the p53 gene was analyzed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) and polymerase chain reaction-heteroduplex DNA (PCR-HET), and the mutations were subsequently confirmed by DNA sequencing. Moderate-to-significant genomic instability was detected in all leukoplakias analysed. Nine unique amplicons, present in leukoplakias but not in normal tissue, were retrieved and successfully characterized. The p53 gene was mutated in 40.6% of patients. Four patients with moderate instability and mutated p53 developed OSCC. The data obtained in this study support and concretize the thesis that premalignant lesions possess many of the alterations found in cancer before the development of a malignant phenotype. Inactivation or mutation of the p53 tumor-suppressor might be an early event contributing to genomic instability and increasing the risk of malignant transformation.

High frequency of p16 and p14 promoter hypermethylation and marked telomere instability in salivary gland tumors.

OBJECTIVES to investigate p16(INK4a) and p14(ARF) tumor suppressor gene methylation status, determine telomere length and assess the importance of these epigenetic and genetic parameters in the development of pleomorphic adenoma and carcinoma ex pleomorphic adenoma of the parotid salivary glands. MATERIALS AND METHODS Genomic DNA from paraffin-embedded samples of 50 pleomorphic adenomas and 10 carcinomas ex pleomorphic adenoma was subjected to methylation specific polymerase chain reaction for hypermethylation analyses and real time polymerase chain reaction for the relative telomere length calculations. RESULTS Promoter hypermethylation of the two genes was a very frequent event in both neoplasms - between 60% and 90% of samples were hypermethylated - but without significant difference between the groups. The mean relative telomere length in the pleomorphic adenoma group was significantly increased in comparison to the control group (P=0.00), and significantly decreased in comparison to the carcinoma group (P=0.05). Telomeres were also longer in myxoid and cellular histological subtypes of adenomas than in the classic type (P=0.044 and P=0.018, respectively). Longer telomeres were more frequent in tumors with hypermethylated p14(ARF) alleles (P=0.013). CONCLUSION Promoter hypermethylations seems to be an important mechanism of p16(INK4a) and p14(ARF) inactivation in parotid gland tumors. Telomeric lengthening appears to be involved in the pathogenesis of both benign and malignant tumors of the parotid glands.

The amplification of c-erb-B2 in cancer-free surgical margins is a predictor of poor outcome in oral squamous cell carcinoma

The tumour subtype, TNM classification, and histopathological data are sometimes not sufficient for understanding and assessing the behaviour of oral cancers. In an attempt to find additional markers of tumour biology and behaviour, this study sought to determine the incidence and consequently the relevance of c-erb-B2, c-Myc, and H-ras gene alterations in tumour-free margins of oral squamous cell carcinoma (OSCC). Fifty samples of OSCC were analyzed for c-erb-B2 and c-Myc amplification by real-time polymerase chain reaction and for H-ras point mutations by sequencing. A relatively high incidence of genetic lesions was detected: 22% of cases had c-erb-B2 and 30% had c-Myc amplification, whilst only 12% harboured H-ras mutations. Kaplan-Meier analysis and the log-rank test showed statistically significant differences in 5-year survival rates and relapse between patients with tumour margins positive for c-erb-B2 amplification and those with margins that were negative (P=0.002). H-ras and c-Myc alterations could not be associated with tumour behaviour. Molecular analysis of margins, targeting cancer genes, could identify additional, independent predictors of risk and outcome in OSCC.

Somatic Mutation and Polymorphism Analysis in Pleomorphic Adenomas of the Salivary Glands / SOMATSKE MUTACIJE I ANALIZA POLIMORFIZAMA U PLEOMORFNIM ADENOMIMA PLJUVAČNIH ŽLEZDA

Summary Background: Genetic studies of salivary gland neoplasms were mainly focused on chromosomal changes, and some specific patterns of chromosome translocations have been described. However, molecular alterations, in particular the role of HER-2/H-ras/c-myc signalling cascade in pleomor- phic adenoma pathogenesis (PA), are less well characterized. In addition, data on single nucleotide polymorphisms (SNPs) as potential susceptibility factors for PA development are also quite scarce. Methods: Mutational analyses were performed by means of real-time PCR (HER-2 and c-myc amplification analysis), PCR-SSCP and sequencing (H-ras point mutation detec- tion). Polymorphisms analysis was performed by PCR-RFLP (survivin and MMP-9 genes). Results: Amplification of HER-2 and c-myc has been found in 13% and 9% of PA cases respectively. Point mutations in H-ras codons 12/13 have been detected in 17% of PAs. No correlation could be established between these alterations and clinical characteristics of PAs, whereas they might play a role in a subset of malignant salivary gland tumours. As for survivin -31 G/C polymorphism, C allele carriers had a 4-fold decrease of the risk of developing PA (p=0.05). Carriers of the variant allele T of the -1562C/T SNP in MMP-9 gene had a 4-fold increase of the risk of developing PA (p<0.001). Conclusions: A longer follow-up of PA patients harbouring mutations could uncover a prognostic role of HER-2 and c- myc amplification as predictors of adenoma transformation into carcinoma. Both survivin and MMP-9 promoter poly- morphisms represent susceptibility factors for the develop- ment of PAs in the Serbian population. Kratak sadržaj Uvod U ispitivanjima mehanizama nastanka tumora plju- vačnih žlezda uglavnom dominiraju citogenetičke študije, pa su tako detektovane i opisane različite hromozomske trans- lokacije sa specifičnim obrascem javljanja. Medutim, mole- kularne promene u ovim tumorima i dalje su relativno slabo poznate, a pogotovo je malo podataka o potencijalnom značaju signalnog puta HER-2/H-ras/c-myc u razvoju i pro- gresiji pleomorfnih adenoma (PA). Takode, retki su i podaci o polimorfizmima pojedinačnog nukleotida (SNP) kao fakto- ra predispozicije za nastanak PA. Metode: Analize somatskih mutacija uradene su metodama real-time PCR (analiza amplifikacije HER-2 i c-myc), PCR-SSCP 1 sekvenciranja (detekcija tačkastih mutacija u H-ras). Analiza polimorfizama pojedinačnih nukleotida (SNP) vršena je prime- nom metode PCR-RFLP (u genima za survivin i MMP-9). Rezultati: Amplifikacija gena HER-2 detektovana je u 13%, c-myc u 9% a tačkaste mutacije u kodonima 12/13 H-ras gena u 17% uzoraka. Nije ustanovljena veza izmedu ovih promena i kliničkih odlika adenoma. Na malom uzorku kar- cinoma, pokazano je da je amplifikacija HER-2 povezana sa recidivima tumora. Nosioci C alela u -31G/C SNP gena za survivin imaju četiri puta manji rizik od nastanka PA (p=0,05), dok nosioci varijantnog alela T kod -1562 C/T SNP u MMP-9 genu imaju četiri puta veči rizik da obole od PA u odnosu na kontrolu (p<0,001). Zaključak: Dužim praćenjem pacijenata sa PA mogla bi da se ustanovi prognostická uloga HER-2 i c-myc amplifikacija kao indikatora za transformaciju adenoma u karcinom. Polimorfizmi u promotorima gena za survivin i MMP-9 pred- stavljaju modulátore rizika za razvoj pleomorfnih adenoma u srpskoj populaciji.

TP53 and C-MYC Co-Alterations – A Hallmark of Oral Cancer Progression / SIMULTANA ALTERACIJA TP53 I C-MYC GENA – OBELEŽJE PROGRESIJE ORALNIH KARCINOMA

Summary Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth most common cancer worldwide. Despite advances in surgery and treatment, the 5-year survival rate has not improved significantly. There- fore, reliable molecular markers for oral cancer progression are badly needed. Methods: We conducted a copy number analysis to esti- mate amplification status of c-myc, cycD1 and EGFR onco- genes, mutational PCR-SSCP analysis to determine activa- tion of H-ras oncogene and inactivation of TP55 tumour suppressor gene and methylation specific PCR analysis to evaluate hypermethylation of p16 and MGMT genes. Results: c-myc oncogene was amplified in 56.7%, cycD1 in 20% and EGFR in 16.7% of Oral Squamous Cell Carci- noma (OSCC) cases while H-ras was activated in 33.3% of samples. Amplification of c-myc was significantly associat- ed with the tumour grade 2. Interestingly, EGFR and H-ras alterations were mutually exclusive. p16 and MGMT were inactivated by hypermethylation in 30% and 13.3% of cases. Co-alteration of cycD1 and p16 were not observed in any of the analyzed samples. TP53 was inactivated in 56.7% of samples and was significantly associated with progression of OSCC, grade 2 and stage 2. Moreover, TP55 and c-myc oncogene were simultaneously altered in grade 2 OSCC. Conclusions: The most promising marker of OSCC pro- gression remains the TP53 tumour suppressor, which is the most frequently mutated gene in oral cancers. Since there is synergism between TP55 and c-myc, it seems that co- alteration of these two genes could be also a good marker of OSCC progression from gradel to grade 2 tumours. Kratak sadržaj Uvod: Skvamocelularni karcinomi glave i vrata (HNSCC) uključujud i skvamocelularni karcinom usne duplje (OSCC) ubrajaju se u šest najčesdh tipova humanih maligniteta. Uprkos značajnim napredcima u hirurškom i terapijskom tretmanu, stopa petogodišnjeg preživljavanja kod ovog tipa maligniteta nije značajnije popravljena. Upravo zato, defin- isanje pouzdanih molekularjnih markera progresije kod OSSC predstavlja apsolutni prioritetet. Metode: Amplifikacioni status c-myc, cycD1 i EGFR gena od reden je pomoču eseja za detekciju broja genskih kopi- ja, aktivacija H-ras onkogena i inaktivacija TP53 tumor supresora odredena je PCR-SSCP mutacionom analizom, a hipermetilacija promotora p16 i MGMT gena je ispitana metil specifičnim PCR-om (MSP). Rezultati: Amplifikacija c-myc onkogena detektovana je kod 56,7%, cycD1 onkogena kod 20%, a EGFR onkogena kod 16,7% analiziranih oralnih skvamocelularnih carcino- ma. Istovremeno, mutaciona aktivacija H-ras onkogena detektovana je kod 33,3% ispitanih uzoraka. Amplifikovani c-myc, statisticki značajno korelira sa gradusom 2 OSCC. Posebno intrigantan je bio nalaz po kom se onkogene akti- vacije u EGFR i H-ras genu medjusobno isključuju. Hipermetilacija promotora p16 gena detektovana je kod 30%, a MGMT kod 13,3% analiziranih uzoraka. Ko-altera- cije cycD1 i p16 gena nisu zapažene ni u jednom od ana- liziranih uzoraka. Inaktivacija TP53 gena detektovana je kod 56,7% uzoraka i utvrdeno je da statistički značajno korelira sa gradusom 2 i statusom 2 OSCC. Pored ovoga, utvrdeno je da statistički značajan broj uzoraka gradusa 2, sa aktiviranim TP53 genom ima istovremeno aktiviran i c-myc onkogen. Zaključak: TP53, najčešče mutirani gen u oralnim karcino- mima, ostaje za sada i najpouzdaniji marker progresije kod OSCC. Obzirom na detektovani sinergizam izmedu TP53 i c-myc gena, možemo reči da su istovremene promene u ova dva gena još pouzdaniji pokazatelj progresije OSSC iz gradusa 1 u gradus 2.