La neoplasia Blastica di derivazione dagli elementi Dendritici Plasmacitoidi è una rara ed aggressiva neoplasia ematologica con una prognosi sfavorevole. Non esiste un regime terapeutico standard di prima linea per questi pazienti e solo pochi studi hanno finora esplorato la genetica del BPDCN mostrando un cariotipo complesso e alterazioni genetiche sporadiche. Lo scopo del nostro studio è stato quello di indagare, mediante sequenziamento massivo, lo stato mutazionale di tutto il genoma codificante di 15 casi di BPDCN e della linea cellulare CAL-1. Sulla base di questi dati, abbiamo seguito un approccio di target sequencing per validare, mediante la piattaforma Miseq, mutazioni in 36 geni selezionati. Il WES ha rivelato 199 SNV ricorrenti in 89 geni, tutti legati a condizioni patologiche. I geni più ricorrentemente mutati tra i campioni sono stati ASXL1 e TET2, due regolatori epigenetici, che giocano un ruolo cruciale nella metilazione del DNA e degli istoni e il loro danno porta ad alterazione della struttura della cromatina. Abbiamo quindi deciso di usare i nostri dati di sequenziamento per esplorare il rimodellamento della cromatina nel BPDCN, guardando lo stato mutazionale di tutti i geni coinvolti nella regolazione epigenetica del DNA. Abbiamo trovato 26 geni modificatori epigenetici mutati in quasi tutti i campioni, sei di questi colpiti da mutazioni stop-gain che hanno portato, presumibilmente, ad una perdita funzionale di attività. Partendo proprio da questo punto abbiamo deciso di procedere con la sperimentazione in vivo creando dei modelli murini somministrando 5-Azacitdina, Decitabina e Romidepsina da soli e in combinazione. Inoltre basandoci su studi pregressi abbiamo anche deciso di trattare i topi con Bortezomib. Queste sperimentazioni hanno portato a risultati incoraggianti, specialmente per il trattamento combinato dei topi con Decitabina e Azacitidina che ha portato a riduzione della massa tumorale e maggiore sopravvivenza dei topi trattati rispetto ad i controlli non trattati. / Blastic plasmacytoid dendritic cell neoplasm is a rare but aggressive hematologic malignancy with a poor prognosis. There is no first-line standard treatment regimen established for patients with BPDCN and only a few studies explored the genetics of BPDCN showing a complex karyotype and sporadic genetic defects. The aim of our study was to investigate, using deep sequencing, the mutational status of all BPDCN coding genome in 15 BPDCN cases and the CAL-1 cell line. Based on these data, we designed a resequencing approach to identify mutations in 36 selected genes with Miseq platform. WES revealed 199 recurrent SNV (at least 2/16 cases) in 89 genes, all related to pathological conditions. The most recurrent mutated genes, among samples, were ASXL1 and TET2, two epigenetic regulators, that play a crucial role in DNA and histones methylation and their damage alter the chromatin structure. We thus decided to use our exome sequencing data to explore the chromatin remodelling of BPDCN, interrogating the mutational status of all the genes involved into the epigenetic regulation of DNA. We found 26 epigenetic modifier genes mutated in almost all samples and six of them are affected by stop gain mutations presumably leading to a functional loss of chromatin remodelling activity. Starting from this point, we decided to proceed with in vivo testing by creating mouse models by administering 5-Azacitdina, Decitabine and Romidepsine alone and in combination. In addition, we are relying on previous studies we have also decided to treat the mice with Bortezomib. These experiments have led to encouraging results, especially for the combined treatment of mice with Decitabine and Azacitidine, which led to tumor shrinkage and increased survival of treated mice compared to the untreated controls.

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. The second part of this work is the clinical, molecular and functional description of a paradigmatic case of primary refractory Burkitt lymphoma characterized by spatial intratumor heterogeneity for the TP53 mutational status, high expression levels of genomic instability and DDR activation markers, primary resistance to chemotherapy and exquisite sensitivity to DDR inhibitors.

Peripheral T-cell lymphomas-not otherwise specified (PTCL/NOS) are the most common T-cell neoplasms. This study sought to reshape the PTCL/NOS sub-classification (including its two main morphological variants, Lennert lymphoma, LL, and Follicular variant, F-PTCL) based on the correspondence between their molecular features and those of different functional T-cell subsets, also assessing the clinical impact of such an approach. We found that PTCLs/NOS could be divided into groups corresponding to T-cell subsets differently reliant on transcription regulators including mTOR and FOXP3, and identified minimal gene sets discriminating among these groups. Notably, by grouping tumors according to their dependency on master regulators of T-lymphocyte fate, we identified three groups (T-cytotoxic, Treg/TFH, and other-T-helper) characterized by specific genetic patterns and significantly different clinical outcomes. Immunohistochemistry partially substituted for the molecular analysis by consistently recognizing only Treg and TFH cases. Finally, targeted inhibition of MTOR in T-helper cases (that were characterized by genetic lesions targeting the pathway) was proved to be effective ex vivo. We conclude that PTCL/NOS can be divided into subgroups corresponding to different cellular counterparts, characterized by different genetic patterns and possibly sensitivity to specific therapeutic approaches. Furthermore, we identified different gene and microRNA signatures for LL capable of differentiating it from other PTCL/NOS and enriched in cytotoxic function. Moreover, PI3K/Akt/mTOR pathway emerged as novel therapeutic targets for LL. Additionally, LL showed some differences with other PTCL/NOS in terms of clinical features, all supporting its recognition as a distinct entity. Besides, we found that F-PTCL has a distinct molecular signature more similar to PTCL/NOS rather than AITL, and therefore cannot be included among AITLs at least based on GEP, although this necessities more genetic studies. Overall, these results may impact on PTCL classification as well as on future studies aimed to define the more appropriate therapeutic strategy for each identified subgroup/entity.

Il carcinoma squamoso orale (CSO) è spesso preceduto da lesioni definite potenzialmente maligne tra cui la leucoplachia e il lichen ma una diagnosi precoce avviene ancora oggi in meno della metà dei casi. Inoltre spesso un paziente trattato per CSO svilupperà secondi tumori. Scopo del lavoro di ricerca è stato: 1) Studiare, mediante metodica di next generation sequencing, lo stato di metilazione di un gruppo di geni a partire da prelievi brushing del cavo orale al fine di identificare CSO o lesioni ad alto rischio di trasformazione maligna. 2) Valurare la relazione esistente tra sovraespressione di p16INK4A e presenza di HPV in 35 pazienti affetti da lichen 3) Valutare la presenza di marker istopatologici predittivi di comparsa di seconde manifestazioni tumorali 4) valutare la relazione clonale tra tumore primitivo e metastasi linfonodale in 8 pazienti mediante 2 metodiche di clonalità differenti: l’analisi di mtDNA e delle mutazioni del gene TP53. I risultati hanno mostrato: 1) i geni ZAP70 e GP1BB hanno presentato un alterato stato di metilazione rispettivamente nel 100% e nel 90,9% di CSO e lesioni ad alto rischio, mentre non sono risultati metilati nei controlli sani; ipotizzando un ruolo come potenziali marcatori per la diagnosi precoce nel CSO. 2)Una sovraespressione di p16INK4A è risultata in 26/35 pazienti affetti da lichen ma HPV-DNA è stato identificato in soli 4 campioni. Nessuna relazione sembra essere tra sovraespressione di p16INK4A e virus HPV. 3)L’invasione perineurale è risultato un marker predittivo della comparsa di recidiva locale e metastasi linfonodale, mentre lo stato dei margini chirurgici si è rilevato un fattore predittivo per la comparsa di secondi tumori primitivi 4) Un totale accordo nei risultati c’è stato tra analisi di mtDNA e analisi di TP53 e le due metodiche hanno identificato la presenza di 4 metastasi linfonodali non clonalmente correlate al tumore primitivo. / Oral Squamous Cell Carcinoma (OSCC) is often preceded by Potentially Malignant Lesions, Leukoplakia and Lichen Planus (LP), but still today is usually diagnosed in advanced stage. Additionally patients treated for OSCC have a good chance to develop a second primary OSCC. The purpose of the research was: 1) to assess, by bisulfite Next Generation Sequencing, the methylation status of a list of candidate genes obtained from oral brushing specimens to early detect OSCC using non-invasive procedures. 2) to yield more insights into the relationship between p16INK4A over-expression and the presence of HPV-DNA in 35 LP samples. 3) to identify the clinicopathologic parameters significantly related to a second loco-regional event in a population of 180 patients treated for primary OSCC 4) to evaluate the relationship between primary OSCC and lymph node metastasis in 8 patients using two different 2 clonality tests: mt-DNA D-loop and TP53 sequence analysis. The results showed that: 1) ZAP70 and GP1BB showed an aberrant methylation status respectively in 100% and 90,9% of OSCC and High Risk lesions, while resulted unmethylated in normal healthy donors. DNA methylation analysis of GP1BB and ZAP70 seems to be a promising non invasive tool to early detect OSCC from oral brushing specimens. 2) p16INK4A over-expression was found in 26/35 LP samples, whereas HPV was found in 4/35 lesions: 3 low-risk HPV and 1 high-risk HPV. P16INK4A overexpression is not correlated with HPV in patients with LP. 3) Perineural invasion was significantly related to lymph-node metastasis (18% vs 8%) and Local Recurrence (15% vs 5%), while the status of the surgical margin resulted an independent factor influencing Second Primary Tumor. 4) The results from mtDNA analysis showed a good agreement with results of p53 mutation analysis and revealed the presence of 4/11 neck nodal metastases not phylogenetically related respect to primary tumor.

Fifty-two cases of monomorphic post-transplant lymphoproliferative disorders (M-PTLD), developed in patients undergone solid organ or bone marrow transplantation, were studied by the application of the tissue micro-array (TMA) technology. They included 50 cases of diffuse large B-cell lymphomas (DLBCL) and 2 Burkitt lymphomas (BL). In order to evaluate the immune-profile a large panel of antibodies was applied including several new markers (Cyclin D2, Cyclin D3, p27, PKC-β, FOXP-1 and Survivin) identified as negative prognostic factors in DLBCL of the immunocompetent patient. Out of 50 DLBCL, 23 cases (46%) had an Activated B Cell (ABC) phenotype, 8 (16%) a Germinal Centre B-cell (GCB) phenotype, and 11 (22%) an Unclassified (UC) phenotype. In 8 cases (16%) the subtype was not demonstrable due to sub-optimal preservation or loss of the tissue core. FISH analysis detected BCL2 gene amplification and MYC rearrangement. EBV was identified in 32 cases (64%) performing immunohistochemistry (LMP-1) and in situ hybridization (EBER). Clinical data and follow-up were available in all cases of malignant lymphomas but one. Thirty-two patients died for progression of disease or complications related to transplant (bleeding, bacterial infections, and multi-organ failure); 17 patients are actually alive and disease-free. M-PTLD are aggressive lymphomas characterized by very poor outcome. The neoplastic process is stimulated by a prolonged immunosuppressive status which is capable to induce alterations of the immune system and allow EBV reactivation in previously infected patients. Indeed EBV infection seems to be the most significant risk factor to predict the development of a PTLD while age, sex, site of involvement and type of transplant do not have significant correlation. Furthermore DLBCL arisen in a setting of immunodeficiency share phenotypic and molecular features with DLBCL of the immunocompetent patient. In particular, the former shows a high incidence of BCL2 gene amplification and this aberration typically correlates with “non-GCB” phenotype. Also M-PTLD do express prognostic markers (PKC-β, cyclin D2, FOXP-1, and Survivin): notably, in our study, PKC-β and FOXP-1 were frequently expressed and they were predictive of a shorter overall survival even in lymphomas recognized to have a good prognosis (GCB-type). Given the fact that such molecules are detectable at the time of the diagnosis, we postulate whether a “tailored” or more specific therapy might be applied in the management of the immune-compromised patient.

No description available.

Recent reports showed that early-interim PET-scan is the only tool predicting treatment outcome in advanced-stage classical Hodgkin lymphoma (asCHL). We evaluated the prognostic impact of a series of immunohistochemical markers, mentioned in literature as prognostic factors, on tissue microarrays assembled from biopsies of 220 patients: STAT1, SAP, TOP2A, PCNA and CD20, both in neoplastic (HRSC) and microenvironment cells (MC); RRM2, MAD2, CDC2, BCL2, P53, BCL11A and EBER in HRSC; ALDH1A1, TIA-1, granzyme B, perforin, FOXP3, and PD-1 in MC. All patients had been treated with standard ABVD ± Rx therapy. Interim-PET after 2 ABVD courses was evaluated according to the criteria indicated by Gallamini in his study (Journal of Clinical Oncology, 2007). The survival analysis has been performed in a subset of 138 patients whose complete clinical information were available: the mean age was 33.3 years (14-79), the stage III-IVB in 98 and IIB in 40, and the mean follow-up 38.1 months (7.6-71.9). Histopathology review showed: NS-I 75, NS-II 22, MC 20, DL 3, and CHL/nos 18 cases. Interim-PET was positive in 30 patients, while treatment failure was recorded in 32. In univariate analysis the factors related to treatment outcome were BCL2 on HRSC (cut-off value 50%), STAT1/SAP on MC, and PET (Log-rank 6.9, 7.9 and 93.9 respectively). The combined expression of STAT1 and SAP was scored in three levels depending on the architectural pattern: score 0 for expression of both with a diffuse/rosetting pattern; score 1 for discordant combination of diffuse/rosetting and scattered patterns; score 2 for both markers with a scattered pattern; the 3y-PFS were 87.4%, 69.9% and 61.9% respectively. In multivariate analysis PET, BCL2 and STAT1/SAP remained significant (HR: 24.8, 4.6, 7.5 and 5.6, respectively; p<.01). The proposed model is able to predict treatment response in AsCHL, even if with a lower efficacy than PET. However, unlike PET, it can be applied upfront therapy.

Gliomas are the most common primary brain tumours. Despite advances in surgical techniques, postoperative supportive care, radiation and adjuvant systemic therapy, the life expectancy of patients with high grade glioma has remained essentially poor. Furthermore differential diagnosis among astrocytomas, oligodendrogliomas and oligoastrocytomas is very challenging and subject to inter-observer variability. The purpose of the research was: 1) to investigate a series of high grade and low grade gliomas at gene and protein (immunohistochemistry) levels to disclose possible genetic portraits of malignancy; 2) to verify the utility of Nogo-A, Olig-2 and synaptophysin in providing a correct histological diagnosis of oligodendroglioma and to investigate a possible complementary role in selecting the best areas suitable for detecting 1p/19q codeletion using FISH analysis; 3) to study the role of microRNA in high grade gliomas. In order to obtain these goals large series of brain tumors were studied with DNA microarrays, immunohistochemistry and RT-PCR The results demonstrated that: - Overexpression of IGFBP-2 and CDC20 is highly related to glioblastomas and their immunopositivity can be useful for the identification of glioblastoma in small biopsies. - Nogo-A is the most useful and specific marker in differentiating oigodendrogliomas from other gliomas. Furthermore, using a Nogo-A driven FISH analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas. - microRNAs can be studied in paraffin embedded tissues better than in fresh tissues. A series of six microRNA, significatively deregulated in glioblastomas, may represent a genetic signature with prognostic and predictive value and could constitute candidates for novel anti-cancer therapeutics.

No description available.

I linfomi a cellule T periferiche rappresentano circa il 12% di tutte le neoplasie linfoidi.In questo studio, abbiamo effettuato un’analisi di miRNA profiling (TaqMan Array MicroRNA Cards A) su 60 campioni FFPE suddivisi in: PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) e cellule T normali (N=13). Abbiamo identificato 4 miRNA differenzialmente espressi tra PTCLs e cellule T normali. Inoltre, abbiamo identificato tre set di mirna che discriminano le tre entita di PTCLs nodali / AIMs: Here, we performed an extensive miRNA profiling of PTCLs in order to identify differentially expressed miRNA, either involved in their pathogenesis or potentially useful for their differential diagnosis. Methods: We studied by miRNA profiling (TaqMan Array MicroRNA Cards A) 60 samples including PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) and normal T cells (N=13); in addition, 40 independent PTCL cases were studied by qRT-PCR as validation. We assess a GEP and miRNA Profiling. Findings: we identified 256 miRNA differentiating the two groups. In conclusion, miRNA profiling allowed to identify miRNA possibly involved in PTCL pathogenesis and to develop a novel diagnostic tool for the differential diagnosis of the commonest subtypes.