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Aberrant regulation of the cell cycle is a typical feature of all forms of cancer. In head and neck squamous cell carcinoma (HNSCC), it is often associated with the overexpression of cyclin D1 (CCND1). However, it remains unclear how CCND1 expression changes between tumor and normal tissues and whether human papillomavirus (HPV) affects differential CCND1 expression. Here, we evaluated the expression of D-type cyclins in a cohort of 94 HNSCC patients of which 82 were subjected to whole genome expression profiling of primary tumors and paired normal mucosa. Comparative analysis of paired samples showed that CCND1 was upregulated in 18% of HNSCC tumors. Counterintuitively, CCND1 was downregulated in 23% of carcinomas, more frequently in HPV-positive samples. There was no correlation between the change in D-type cyclin expression and patient survival. Intriguingly, among the tumors with downregulated CCND1, one-third showed an increase in cyclin D2 (CCND2) expression. On the other hand, one-third of tumors with upregulated CCND1 showed a decrease in CCND2. Collectively, we have shown that CCND1 was frequently downregulated in HNSCC tumors. Furthermore, regardless of the HPV status, our data suggested that a change in CCND1 expression was alleviated by a compensatory change in CCND2 expression.

Background. Acute lymphoblastic leukemia (ALL) is the most frequent neoplastic disease in children and it is generally characterized by favorable outcome, with the long-term cure rate approaching 90%. The stratification of patients into risk groups allows a more specific risk-adapted treatment, saving patients with a low risk of relapse from excessive toxicity, while improving outcome in patients with a high risk of relapse by intensive treatment. Risk stratification has been based on clinical features and response to treatment. NOTCH1 activation and PTEN inactivation are common events implied in leukemogenesis, and appear attractive candidates as drivers for risk stratification. NOTCH1 is involved in transcriptional upregulation of IL7R, a gene known to be mutated in 10% of T-ALL cases. In addition, the NOTCH1 signaling is also involved in the regulation of cyclin D3. This protein is involved in cell proliferation and it is necessary for T-ALL development. Given the fact, in recent studies new genetic alterations are associated with prognosis and could be used for patient's stratification, as well as for targeted therapy. All these highlight the import of the identification of new mutations that could play a pivotal role in the leukemogenesis. Aim of the study. The purpose of this study was to evaluate the prognostic significance NOTCH1 and PTEN alterations in childhood T-ALL. To this purpose, a large pediatric T-ALL patient cohort treated according to the AIEOP ALL2000/R2006 and AIEOP ALL2009 protocols was comprehensively characterized with respect to NOTCH1 and PTEN; the NOTCH1/PTEN mutational status were then correlated with the cumulative incidence of relapse and event free survival. Using the somatic mutations as specific genetic markers for tumor cell we applied next-generation ultra-deep sequencing technologies to detect the presence of residual cells in the bone marrow samples during therapy is the second aim of this study. Therefore, the mutational status of IL7R at diagnosis and +33 day therapy was characterized and it was then correlated with MRD RQ-PCR. Finally, it was analyzed the CCND3 mutation recurrence to identify new genes involved in leukemogenesis. Materials and methods. Bone marrow samples from 272 children with T-ALL obtained at the time of first diagnosis were analyzed with Sanger sequencing for PTEN mutations. Activating NOTCH1 mutations were detected by Sanger sequencing of the exons coding for the heterodimerisation (HD), PEST and TAD domains. Activating IL7R mutations were analyzed by Sanger sequencing and deep-amplicon sequencing of 227 T-ALL patients. The exome from 8 children with T-ALL diagnosis high-risk and related control was analyzed by Ion Torrent Technology. Finally, CCND3 mutations were analyzed in 80 T-ALL patients at diagnosis by Sanger sequencing. Results. The frequency of PTEN and NOTCH1 alterations was 12% and 65% respectively. The survival analysis on the total cohort on T-ALL patients shows a higher incidence of relapse and a lower survival in patients with inactivating PTEN mutations. Kaplan-Meyer estimate of event free survival (EFS) show a positive prognostic significance of NOTCH1 mutations in AIEOP T-ALL total cohort, but this result is not confirmed in the last therapeutic protocol analyzed AIEOP ALL2009. Because the only detectable difference between this cohort and those described in the literature is the treatment that has been applied, we hypothesize that the effect of NOTCH1 and PTEN mutational status on the risk of relapse depends on the treatment applied. By deep-sequencing the IL7R clonal mutations have been identify with great sensitivity, even if the frequency of the mutation was less than 10%. Finally, the Ion Proton System technology identified a missense mutation in CCND3 gene, this mutation have shown a recurrence 6% within a larger cohort of T-ALL by Sanger sequencing. Conclusions. In the patient cohort that has been analyzed, the PTEN mutation were correlated with outcome and were generally important for patients stratification in high risk groups; in contrast, the NOTCH1 mutation correlation on outcome depends on the type of therapeutic treatment adopted and is not generally useful for risk stratification. Future models of risk stratification will need to include more genetic markers. Preliminary results of IL7R show that ultra deep-sequencing have allow detecting mutations at subcloanl level and it was possible follow the leukemic clone with the IL7R mutation during therapy. Finally, the identification of somatic mutations and the expression of the CCND3 gene in onset blasts suggests that these genetic alterations could help the disease progression. The future functional studies will need to evaluate the possible gain-of-function of the mutated protein in leukemic blasts and to provide a new therapeutic target.

Cyclin D3 and CDK11 are downregulated in pancreatic islet endocrine cells during the autoimmune attack progression in autoimmune-prone NOD (Non-obese diabetic) mouse strain. D-type cyclins are crucial in order to connect mitogenic signals with the Rb/E2F pathway, which regulates transcription of factors involved in further cell cycle progression. CDK11, protein-kinase PITSLRE, exhibits two gene products: p58 and p110 (p130 in mouse) in humans. CDK11p110 regulates transcription and RNA splicing. CDK11p110 is expressed in all cell cycle phases, while CDK11p58 is only expressed during mitosis (G2/M) and is essential in apoptosis. The interaction between CDK11p58 and cyclin D3 has been reported and it represses certain nuclear receptors action. This observation may suggest that in pancreatic beta cells simultaneous downregulation of cyclin D3 and CDK11 may obey to a coordinated regulation of both molecules. In this thesis we have studied whether there is a causal relationship between coordinated cyclin D3 and CDK11 downregulation and type 1 diabetes in vivo and in vitro.The outcome of our research will allow us to establish whether cyclin D3 and CDK11 are molecular targets in type1 diabetes, La ciclina D3 i CDK11 estan regulades a la baixa en les cèl•lules endocrines de l'illot pancreàtic durant la progressió atac autoimmune en la soca de ratolins NOD propensos a la diabetis autoimmune. Les ciclines de tipus D són crucials per connectar senyals mitogèniques amb la via Rb/E2F , que regula la transcripció de factors implicats en la progressió del cicle cel • lular . El gen que codifica per a la CDK11 , proteïna-quinasa PITSLRE, té dos productes gènics en humans: p58 i p110 ( p130 al ratolí ). LaCDK11p110 regula la transcripció i processament del 'ARN. La CDK11p110 s'expressa en totes les fases del cicle cel•lular , mentre que la CDK11p58 només s'expressa durant la mitosi (G2 / M) i participa en processos apoptòtics . La interacció entre CDK11p58 i ciclina D3 reprimeix certa acció d'alguns receptors nuclears, per exemple afecta negativament l'activitat transcripcional del receptor d'andrògens . Aquesta observació pot suggerir que en les cèl•lules beta del pàncrees la regulació a la baixa simultània de ciclina D3 i CDK11 pot obeir a una regulació coordinada de les dues molècules . En aquesta tesi s'ha estudiat si hi ha una relació causal entre la regulació a la baixa coordinada de ciclina D3 i CDK11 i l'aparició de la diabetis tipus 1 , in vivo i in vitro .El resultat de la nostra recerca ens permetrà establir si la ciclina D3 i la CDK11 podran ser blancs moleculars en la diabetis tipus 1., Ciclina D3 y CDK11 están regulados a la baja en las células endocrinas del islote pancreático durante la progresión ataque autoinmune en la cepa de ratones NOD predispuesta genéticamente a la autoinmunidad. Los ciclinas de tipo D son cruciales para conectar las señales mitogénicas con la vía Rb/E2F, la cual regula la transcripción de factores implicados en la progresión del ciclo celular. El gen que codifica para la CDK11, la proteína-quinasa PITSLRE, tiene dos productos génicos en humanos: p58 y p110 ( p130 en el ratón). CDK11p110 regula la transcripción y procesameinto del ARN. CDK11p110 se expresa en todas las fases del ciclo celular, mientras que CDK11p58 sólo se expresa durante la mitosis (G2 / M) y está implicada en procesos apoptóticos. La interacción entre CDK11p58 y ciclina D3 reprime cierta acción de algunos receptores nucleare, por ejemplo, afecta negativamente a la actividad transcripcional del receptor de andrógenos. Esta observación puede sugerir que en las células beta del páncreas la regulación a la baja simultánea de ciclina D3 y CDK11 puede obedecer a una regulación coordinada de ambas moléculas. En esta tesis se ha estudiado in vivo e in vitro si existe una relación causal entre la regulación a la baja coordinada de ciclina D3 y CDK11 y la aparición de la diabetes tipo 1.El resultado de nuestra investigación nos permitirá establecer ciclina D3 y CDK11 como blancos moleculares en la diabetes tipo 1.