The constitutive activation of the DNA damage response pathway is a novel therapeutic target in aggressive B-cell lymphoma

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.

Histone deacetylase inhibitors in adult patients with diffuse large b-cell lymphoma relapsed/refractory: a phase II study with Panobinostat

Backgrounds:Treatment of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) not eligible to high dose therapy represents an unmet medical need. Panobinostat showed encouraging therapeutic activity in studies conducted in lymphoma cell lines and in vivo in patients with advanced hematologic malignancies.Purpose:FIL-PanAL10 (NCT01523834) is a phase II, prospective multicenter trial of the Fondazione Italiana Linfomi (FIL) to evaluate safety and efficacy of single agent Panobinostat as salvage therapy for R/R DLBCL patients and to evaluate a possible relationships between response and any biological features. Patients and Methods:Patients with R/R DLBCL were included. The treatment plan included 6 induction courses with Panobinostat monotherapy followed by other 6 courses of consolidation. The primary objective was to evaluate Panobinostat activity in terms of overall response (OR); secondary objectives were: CR rate, time to response (TTR), progression-free survival (PFS), safety and feasibility of Panobinostat. We included evaluation of the impact of pharmacogenetics, immunohistochemical patterns and patient’s specific gene expression and mutations as potential predictors of response to Panobinostat as explorative objectives. To this aim a pre-enrollment new tissue biopsy was mandatory. ResultsThirty-five patients, 21 males (60%), were enrolled between June 2011 and March 2014. At the end of induction phase, 7 responses (20%) were observed, including 4 CR (11%), while 28 patients (80%) discontinued treatment due to progressive disease (PD) in 21 (60%) or adverse events in 7 (20%). Median TTR in 9 responders was 2.6 months (range 1.8-12). With a median follow up of 6 months (range 1-34), the estimated 12 months PFS and OS were 27% and 30.5%, respectively. Grade 3-4 thrombocytopenia and neutropenia were the most common toxicities (in 29 (83%) and 12 (34%) patients, respectively. Conclusions The results of this study indicate that Panobinostat might be remarkably active in some patients with R/R DLBCL, showing durable CR

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

Micosi fungoide: indagini strumentali e laboratoristiche

Mycosis fungoides: instrumental and laboratory examinations. Cutaneous lymphomas are a group of disorders characterized by localization of malignant lymphocytes to the skin. Approximately two-thirds of these lymphomas are of T-cell origin. The most common form of cutaneous T-cell lymphoma is mycosis fungoides. Several reviews and guidelines on the management of mycosis fungoides have been published. This study reviews the management and the follow-up of early-stage MF according to our experience.

Analisi molecolare dei linfomi cutanei aggressivi

I linfomi primitivi cutanei riconosciuti nella classificazione della WHO/EORTC si presentano come “entità cliniche distinte” su base clinica, morfologica, immunofenotipica e molecolare. Il fenotipo linfocitario T helper CD4+ caratterizza i CTCL, ma alcune entità a prognosi aggressiva presentano un immunofenotipo citotossico CD8+. Numerosi studi di citogenetica (CGH) e gene-expression profiling (GEP) sono stati condotti negli ultimi anni sui CTCL e sono state riscontrate numerose aberrazioni cromosomiche correlate ai meccanismi di controllo del ciclo cellulare. Scopo del nostro studio è la valutazione delle alterazioni genomiche coinvolte nella tumorigenesi di alcuni CTCL aggressivi: il linfoma extranodale NK/T nasal-type, il linfoma primitivo cutaneo aggressivo epidermotropo (AECTCL) e il gruppo dei PTCL/NOS pleomorfo CD8+. Il materiale bioptico dei pazienti è stato sottoposto alla metodica dell’array-CGH per identificare le anomalie cromosomiche; in alcuni casi di AECTCL è stata applicata la GEP, che evidenzia il profilo di espressione genica delle cellule neoplastiche. I dati ottenuti sono stati valutati in modo statistico, evidenziando le alterazioni cromosomiche comuni significative di ogni entità. In CGH, sono state evidenziate alcune aberrazioni comuni fra le entità studiate, la delezione di 9p21.3, l’amplificazione di 17q, 19p13, 19q13.11-q13.32 , 12q13 e 16p13.3, che determinano la delezione dei geni CDKN2A e CDKN2B e l’attivazione del JAK/STAT signaling pathway. Altre alterazioni definiscono l’amplificazione di c-MYC (8q24) e CCND1/CDK4-6 (11q13). In particolare, sono state evidenziate numerose anomalie genomiche comuni in casi di AECTCL e PTCL/NOS pleomorfo. L’applicazione della GEP in 5 casi di AECTCL ha confermato l’alterata espressione dei geni CDKN2A, JAK3 e STAT6, che potrebbero avere un ruolo diretto nella linfomagenesi. Lo studio di un numero maggiore di casi in GEP e l’introduzione delle nuove indagini molecolari come l’analisi dei miRNA, della whole-exome e whole genome sequences consentiranno di evidenziare alterazioni molecolari correlate con la prognosi, definendo anche nuovi target terapeutici.

Cellular Proliferation in the Prognosis of Intermediate Grade Mast Cell Tumour in Dogs

This was a retrospective study including ninety samples of dogs with a histological diagnosis of intermediate grade cutaneous mast cell tumour (MCT). The objectives of the study were to validate Minichromosome Maintenance Protein 7 (MCM7) as a prognostic marker in MCTs and to compare the ability of mitotic index (MI), Ki67 and MCM7 to predict outcome. The median survival for the entire population was not reached at 2099 days. The mean survival time was 1708 days. Seventy-two cases were censored after a median follow up of 1136 days and eighteen dogs died for causes related to the MCT after a median of 116 days. For each sample MI, Ki67 and MCM7 were determined. The Receiver Operating Characteristic (ROC) curve was obtained for each prognostic marker to evaluate the performance of the test, expressed as area under the curve, and whether the published threshold value was adequate. Kaplan-Meier and corresponding logrank test for MI, Ki67 and MCM7 as binary variables was highly significant (P<0.0001). Multivariable regression analysis of MI, Ki67 and MCM7 corrected for age and surgical margins indicated that the higher risk of dying of MCT was associated with MCM7 > 0.18 (Hazard Ration [HR] 14.7; P<0.001) followed by MI > 5 (HR 13.9; P<0.001) and Ki67 > 0.018 (HR 8.9; P<0.001). Concluding, the present study confirmed that MCM7 is an excellent prognostic marker in cutaneous MCTs being able to divide Patnaik intermediate grade tumours in two categories with different prognosis. Ki67 was equally good confirming its value as a prognostic marker in intermediate grade MCTs. The mitotic index was extremely specific, but lacked of sensitivity. Interestingly, mitotic index, Ki67 and MCM7 were independent from each other suggesting that their combination would improve their individual prognostic value.

Evaluation of the pathogenetic role of cellular subsets of the Follicular Lymphoma Microenvironment

Follicular lymphoma (FL) is a B cell neoplasm, composed of follicle center cells, that accounts for about 20% of all lymphomas, with the highest incidence reported in the USA and western Europe. FL has been considered a virtually incurable disease, with a high response rate alternated with frequent post-therapy relapses or progression towards more aggressive lymphomas. Due to the extreme variability in outcome, many efforts were made to predict prognosis, the need for therapy, and the likelihood of evolution. Even if clinical scores turned out to be robust and easy to use in clinical practice for patient risk stratification, marked heterogeneity in outcome remains within each group and further insights into the biology of FL are needed. The genome-wide approach underscored the pivotal role of the FL microenvironment in the evolution of the disease. In 2004, a landmark study by Dave et al. first described the microenvironment impact on tumor biology. By gene expression profiling they identified two different immune response signatures, involving T-cells and macrophages which seemed to independently predict FL outcome, but their exact is not completely understood and different studies led to variable results. Subsequently, many workgroups identified in amount and distribution pattern of these different cell subsets features which can impact prognosis, this leading to hypothesizing the use of these parameters as surrogate markers of the molecular signature. We aimed to assess the possible contributions of micro-environmental components to FL transformation or progression, its relevance as a prognostic/predictive tool, and its potential role as an innovative therapeutic target. We used immunohistochemical techniques, focusing specifically on macrophages and T-cells subsets, and then found correlations between the presence, proportions, and distribution of these reactive cells and the clinical outcomes leading to the future development of a reliable tool for upfront risk stratification of patients affected by FL.

Molecular analysis of Papillomavirus-induced cutaneous tumors in equids

Papillomavirus associated tumors are well recognized entities in humans as well as in animals. Here is reviewed the current understanding of human papillomavirus (HPV) associated cancers to better understand the oncogenic mechanisms of Equine papillomavirus (EcPV) and Bovine Papillomavirus (BPV) in horses. In the first part of this study the interactions between Equine papillomavirus 2 (EcPV-2) and cell cycle proteins are discussed. EcPV-2 has been recognized as the cause of genital squamous cell carcinomas (SCCs) in horses, but the exact mechanism of carcinogenesis is not fully understood. The aim of the first part of this study is to assess the expression of cell cycle proteins p53, p16, pRB and Cyclin D1 in a series of equine SCCs and papillomas. Results confirm the role of EcPV-2 in the pathogenesis of genital SCCs. Moreover, in a small subset of ocular SCCs, EcPV-2 was detected for the first time. By immunohistochemistry, p53 was mostly expressed in ocular SCCs with a suprabasal localization. Regarding p16, overexpression was associated with increased mitotic index but not with viral infection. Investigation on pRB and Cyclin D1 proteins did not show significant correlation with other variables. The second part of this study is focused on the carcinogenetic mechanisms of BPV in equine sarcoids. The aim of the second part of this study was to characterize the typical histomorphological features of equine sarcoids, assess the expression of cell cycle proteins and Ki-67 proliferation index. Our results confirm that the typical histological features of sarcoids cannot be used to correctly classify the clinical types. Moreover, in a subset of sarcoids low pRB-Cyclin D1 scores were associated with simultaneous high p16 expression. The Ki-67 proliferation index confirm the low proliferative activity of sarcoids, except for tumors displaying a fascicular pattern. Finally, a subset of sarcoids recurred after excision.

Analysis of efficacy and safety of CAR T-cell therapy in relapsed/refractory lymphomas: current indications and future perspectives

In the last few years, the introduction of chimeric antigen receptor (CAR) T-cell therapy into clinical practice has revolutionized the approach to patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL), whose outcome used to be dismal with median overall survival (OS) of approximately 6 months with standard salvage therapy. At our Institute, we started treating diffuse large B-cell lymphoma (DLBCL) patients with CAR T-cell products in August 2019 and they received either axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) as per regulatory indications. This research project presents the 2-year follow-up of the first 53 treated patients. Our first aim is to investigate the feasibility of this treatment strategy in a real-world setting, although the reimbursement criteria set by the Italian Medicines Agency (Agenzia Italiana del Farmaco, AIFA) are very similar to the inclusion criteria of clinical trials and stricter than those established by the regulatory authorities of many foreign countries. One month after infusion, the ORR was 66% with 19 patients already in CR (38%). Restaging at 3, 6 and 12 months post-infusion shows that early CRs tend to be maintained over time and, moreover, that a considerable number of PRs and a few SDs can improve into a CR. The safety data were consistent with what is reported in the literature; toxicity was generally manageable, largely due to the increasing expertise in handling the specific adverse events related to CAR T-cell therapy. Our results confirms that CAR T-cell therapy is both safe and effective in a real-life setting and that it represents a crucial weapon in a subset of patients who were previously doomed to an inevitably severe prognosis.