Predictive Value of CD8 Expression and FoxP3 Methylation in Nasopharyngeal Carcinoma Patients Treated with Chemoradiotherapy in a Non-endemic Area.

Undifferentiated Nasopharyngeal Carcinoma (UNPC) is associated with Epstein-Barr Virus (EBV) and characterized by an abundant immune infiltrate potentially influencing the prognosis. Thus, we retrospectively assessed the significance of immunosuppression in the UNPC microenvironment as prognostic biomarker of treatment failure in a non-endemic area, and monitored the variation of systemic EBV-specific immunity before and after chemoradiotherapy (CRT). DNA and RNA were extracted from diagnostic biopsies obtained by tumor and adjacent mucosa from 63 consecutive EBV+ UNPC patients who underwent radical CRT. Among these patients 11 relapsed within 2 years. The expression of the EBV-derived UNPC-specific BARF1 gene and several immune-related genes was monitored through quantitative RT-PCR and methylation-specific PCR analyses. Peripheral T cell responses against EBV and BARF1 were measured in 14 patients (7 relapses) through IFN-γ ELISPOT assay. We found significantly higher expression levels of BARF1, CD8, IFN-γ, IDO, PD-L1, and PD-1 in UNPC samples compared to healthy tissues. CD8 expression was significantly reduced in both tumor and healthy tissues in UNPC patients who relapsed within two years. We observed a hypomethylated FOXP3 intron 1 exclusively in relapsed UNPC patients. Finally, we noticed a significant decrease in EBV- and BARF1-specific T-cells after CRT only in relapsing patients. Our data suggest that a high level of immunosuppression (low CD8, hypomethylated FoxP3) in UNPC microenvironment may predict treatment failure and may allow an early identification of patients who could benefit from the addition of immune modulating strategies to improve first line CRT.

NOTCH1 mutations are associated with high CD49d expression in chronic lymphocytic leukemia: link between the NOTCH1 and the NF-κB pathways.

In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10-15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (P<0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

Exploiting a new strategy to induce immunogenic cell death to improve dendritic cell-based vaccines for lymphoma immunotherapy.

Although promising, the clinical benefit provided by dendritic cell (DC)-based vaccines is still limited and the choice of the optimal antigen formulation is still an unresolved issue. We have developed a new DC-based vaccination protocol for aggressive and/or refractory lymphomas which combines the unique features of interferon-conditioned DC (IFN-DC) with highly immunogenic tumor cell lysates (TCL) obtained from lymphoma cells undergoing immunogenic cell death. We show that treatment of mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 9-cis-retinoic acid and IFNα (RA/IFNα) induces early membrane exposure of Calreticulin, HSP70 and 90 together with CD47 down-regulation and enhanced HMGB1 secretion. Consistently, RA/IFNα-treated apoptotic cells and -TCLs were more efficiently phagocytosed by DCs compared to controls. Notably, cytotoxic T cells (CTLs) generated with autologous DCs pulsed with RA/IFNα-TCLs more efficiently recognized and specifically lysed MCL or DLBCL cells or targets loaded with several HLA-A*0201 cyclin D1 or HLA-B*0801 survivin epitopes. These cultures also showed an expansion of Th1 and Th17 cells and an increased Th17/Treg ratio. Moreover, DCs loaded with RA/IFNα-TCLs showed enhanced functional maturation and activation. NOD/SCID mice reconstituted with human peripheral blood lymphocytes and vaccinated with autologous RA/IFNα-TCL loaded-IFN-DCs showed lymphoma-specific T-cell responses and a significant decrease in tumor growth with respect to mice treated with IFN-DC unpulsed or loaded with untreated TCLs. This study demonstrates the feasibility and efficacy of the use of RA/IFNα to generate a highly immunogenic TCL as a suitable tumor antigen formulation for the development of effective anticancer DC-based vaccines.

Improved Natural Killer cell activity and retained anti-tumor CD8(+) T cell responses contribute to the induction of a pathological complete response in HER2-positive breast cancer patients undergoing neoadjuvant chemotherapy.

BACKGROUND: Locally advanced HER2-overexpressing breast cancer (BC) patients achieve a high rate of pathological complete responses (pCR) after neoadjuvant chemotherapy (NC). The apparently unaltered immune proficiency of these patients together with the immune-modulating activities of NC drugs suggest a potential contribution of host immunity in mediating clinical responses. We thus performed an extensive immunomonitoring in locally advanced BC patients undergoing NC to identify immunological correlates of pCR induction. METHODS: The immune profile of 40 HER2-positive and 38 HER2-negative BC patients was characterized at diagnosis and throughout NC (Paclitaxel and Trastuzumab, or Docetaxel and Epirubicin, respectively). The percentages of circulating immune cell subsets including T and B lymphocytes, Natural Killer (NK) cells, regulatory T cells, T helper 17 lymphocytes, were quantified by multiparametric flow cytometry. NK cells functional activity was evaluated through the analysis of NF-kB nuclear translocation by Multispectral flow cytometry, and with the in vitro monitoring of Trastuzumab-mediated antibody-dependent cell cytotoxicity (ADCC). CD8(+) T cell responses against six different tumor-associated antigens (TAA) were characterized by IFN-γ ELISPOT and IFN-γ/IL-2 DualSpot assays. RESULTS: After NC, HER2-positive patients showed a significant increase in the number of NK cells and regulatory T cells irrespective of the pathological response, whereas patients undergoing a pCR disclosed higher percentages of T helper 17 cells. Notably, a significant increase in the number of activated NK cells was observed only in HER2-positive patients achieving a pCR. Characterization of anti-tumor T cell responses highlighted sustained levels of CD8(+) T cells specific for survivin and mammaglobin-A throughout NC in patients undergoing a pCR in both arms. Moreover, HER2-positive patients achieving a pCR were characterized by a multi-epitopic and polyfunctional anti-tumor T cell response, markedly reduced in case of partial response. CONCLUSIONS: These results indicate that maintenance of functional T cell responses against selected antigens and improvement of NK cell proficiency during NC are probably critical requirements for pCR induction, especially in HER2-positive BC patients. Trail registration: TRIAL REGISTRATION NUMBER: NCT02307227, registered on ClinicalTrials.gov ( http://www.clinicaltrials.gov , November 26, 2014).