Davide Brusa

Correlation between NK function and response to trastuzumab in metastatic breast cancer patients.

BackgroundTrastuzumab is a monoclonal antibody selectively directed against Her2 and approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include mediation of antibody-dependent cellular cytotoxicity (ADCC) by triggering FcγRIII on natural killer (NK) cells. This study addresses the correlation between overall NK function and trastuzumabs clinical activity.Subjects and methodsClinical and immunological responses were assessed in 26 patients receiving trastuzumab monotherapy as maintenance management after chemotherapy (8 mg/kg load and then standard doses of 6 mg/kg every 3 weeks). Cytotoxic activity against the MHC class I-negative standard NK target K562 cell line and HER2-specific ADCC against a trastuzumab-coated Her2-positive SKBR3 cell line were assessed in peripheral blood mononuclear cells (PBMC) harvested after the first standard dose. After six months, seventeen patients were scored as responders and nine as non-responders according to the RECIST criteria, while Progression-Free Survival (PFS) was calculated during a 12 months follow-up.ResultsThe responders had significantly higher levels of both NK and ADCC activities (p < 0.05) that were not different from those of eleven normal controls. The NK activity of the non-responders was significantly (p < 0.05) lower than that of the normal controls. At twelve months, there was a marked correlation between PFS and NK activity only. PFS was significantly longer in patients with high levels of NK activity, whereas its pattern was unrelated to high or low ADCC activity.ConclusionOne of the mechanisms of action of trastuzumab is NK cell-mediated ADCC lysis of the Her2-positve target cell. We show here that its potency is correlated with the short-term response to treatment, whereas longer protection against tumor expansion seems to be mediated by pure NK activity.

The PD-1/PD-L1 axis contributes to T cell dysfunction in chronic lymphocytic leukemia

Chronic lymphocytic leukemia is marked by profound defects in T-cell function. Programmed death-1 is a receptor involved in tumor-mediated immunosuppression through binding of the PD-L1 ligand. Multiparametric flow cytometry and immunohistochemistry were used to study PD-1/PD-L1 expression. Functional assays were used to determine the involvement of the PD-1/PD-L1 axis in T-cell responses. PD-1 expression by CD4+ and CD8+ T lymphocytes was significantly higher in 117 chronic lymphocytic leukemia patients than in 33 donors of a comparable age. CD4+ and CD8+ T lymphocytes from chronic lymphocytic leukemia patients displayed increased numbers of effector memory and terminally differentiated cells, respectively, when compared to controls. The number of effector memory CD4+ and terminally differentiated CD8+ lymphocytes positively associated with a more advanced stage of disease, treatment requirements and unfavorable genomic aberrations. Furthermore, leukemic lymphocytes expressed higher levels of PD-L1 than circulating B lymphocytes from normal donors. PD-1 and PD-L1 surface expression spiked in proliferating T and B lymphocytes, suggesting that this interaction works efficiently in activated environments. Within chronic lymphocytic leukemia proliferation centers in the lymph node, CD4+/PD-1+ T lymphocytes were found to be in close contact with PD-L1+ chronic lymphocytic leukemia cells. Lastly, functional experiments using recombinant soluble PD-L1 and blocking antibodies indicated that this axis contributes to the inhibition of IFN-γ production by CD8+ T cells. These observations suggest that pharmacological manipulation of the PD-1/PD-L1 axis may contribute to restoring T-cell functions in the chronic lymphocytic leukemia microenvironment.

CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death

Extracellular adenosine (ADO), generated from ATP or ADP through the concerted action of the ectoenzymes CD39 and CD73, elicits autocrine and paracrine effects mediated by type 1 purinergic receptors. We have tested whether the expression of CD39 and CD73 by chronic lymphocytic leukemia (CLL) cells activates an adenosinergic axis affecting growth and survival. By immunohistochemistry, CD39 is widely expressed in CLL lymph nodes, whereas CD73 is restricted to proliferation centers. CD73 expression is highest on Ki-67(+) CLL cells, adjacent to T lymphocytes, and is further localized to perivascular areas. CD39(+)/CD73(+) CLL cells generate ADO from ADP in a time- and concentration-dependent manner. In peripheral blood, CD73 expression occurs in 97/299 (32%) CLL patients and pairs with CD38 and ZAP-70 expression. CD73-generated extracellular ADO activates type 1 purinergic A2A receptors that are constitutively expressed by CLL cells and that are further elevated in proliferating neoplastic cells. Activation of the ADO receptors increases cytoplasmic cAMP levels, inhibiting chemotaxis and limiting spontaneous drug-induced apoptosis of CLL cells. These data are consistent with the existence of an autocrine adenosinergic loop, and support engraftment of leukemic cells in growth-favorable niches, while simultaneously protecting from the action of chemotherapeutic agents.

PD-L1 marks a subset of melanomas with a shorter overall survival and distinct genetic and morphological characteristics

BACKGROUND Programmed cell death ligand 1 (PD-L1) is a cell surface molecule that plays a critical role in suppressing immune responses, mainly through binding of the PD-1 receptor on T lymphocytes. PD-L1 may be expressed by metastatic melanoma (MM). However, its clinical and biological significance remains unclear. Here, we investigated whether expression of PD-L1 in MM identifies a biologically more aggressive form of the disease, carrying prognostic relevance. PATIENTS AND METHODS PD-L1 expression was analyzed by immunohistochemistry using two different antibodies in primary tumors and paired metastases from 81 melanoma patients treated at a single institution. Protein expression levels were correlated with PD-L1 mRNA, BRAF mutational status and clinical outcome. PD-L1(+) and PD-L1(-) subsets of the A375 cell line were stabilized in vitro and compared using gene expression profiling and functional assays. Results were confirmed using xenograft models. RESULTS PD-L1 membrane positivity was detected in 30/81 (37%) of patients. By multivariate analysis, Breslow thickness and PD-L1 membrane positivity were independent risk factors for melanoma-specific death {PD-L1 5% cutoff [hazard ratio (HR) 3.92, confidence interval (CI) 95% 1.61-9.55 P < 0.003], PD-L1 as continuous variable (HR 1.03, 95% CI 1.02-1.04 P < 0.002)}. PD-L1 expression defined a subset of the BRAF-mutated A375 cell line characterized by a highly invasive phenotype and by enhanced ability to grow in xenograft models. CONCLUSIONS PD-L1 is an independent prognostic marker in melanoma. If confirmed, our clinical and experimental data suggest that PD-L1(+) melanomas should be considered a disease subset with distinct genetic and morpho-phenotypic features, leading to enhanced aggressiveness and invasiveness.

Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.

Post-apoptotic tumors are more palatable to dendritic cells and enhance their antigen cross-presentation activity.

Critical issues for cytotoxic lymphocyte (CTL) cross-priming are (a) the maturation state of dendritic cells (DC), (b) the source of the tumor-associated antigens (TAA) and (c) the context in which they are delivered to DCs. Drug-induced apoptosis has recently been implicated in CTL cross-priming. However, since drug-treatment produces in vivo more tumor cells than the DC default apoptotic clearance program can cope with, they are expected to proceed to secondary necrosis and change their molecular pattern. Here we have addressed this issue on renal carcinoma cells (RCC) by using different apoptotic stimuli. UVC, but not gamma-irradiation or anthracyclins, induced after 4h treatment of the RCC cell line K1 a combination of apoptotic (phosphatydilserine and calreticulin plasma membrane mobilization) and necrotic (membrane incompetence) features. Heat shock protein (Hsp)-70 and chromatin-bound high mobility box 1 HMGB1 protein, typical of necrosis, were released during the further 20h and thus made accessible to co-cultured monocyte-derived immature (i) DC. UVC-treated, secondary necrotic RCC cell lines were cross-presented with higher efficiency by cytokine-matured (m) DC than their early apoptotic (i.e. gamma-irradiated) counterpart. Upstream events such as increased tumor uptake, activation of genes involved in the antigen-processing machinery, and increased expression of costimulatory and maturation molecules were also observed after loading iDC with secondary necrotic, but not apoptotic, tumor cells. These data offer a description of the molecular and immunogenic characteristics of post-apoptotic tumors which can be exploited to increase the efficiency of in vivo and ex vivo TAA delivery to the DC cross-presentation pathway.

The status of PD-L1 and tumor-infiltrating immune cells predict resistance and poor prognosis in BRAFi-treated melanoma patients harboring mutant BRAFV600

BACKGROUND BRAF inhibitors (BRAFi) improve survival in metastatic melanoma patients (MMP) but the duration of clinical benefit is limited by development of drug resistance. Here, we investigated whether the expression of programmed death-ligand 1 (PD-L1) and the density of tumor-infiltrating mononuclear cells (TIMC) predict the occurrence of resistance, hence affecting the clinical outcome in BRAFi-treated MMP. METHODS PD-L1 expression (cutoff 5%) was analyzed by immunohistochemistry with two different antibodies in BRAF(V600)-mutated formalin-fixed and paraffin-embedded samples from 80 consecutive MMP treated with BRAFi at a single institution. TIMC were evaluated by conventional hematoxylin and eosin staining. RESULTS Forty-six and 34 patients received vemurafenib and dabrafenib, respectively. Membranous expression of PD-L1 was detected in 28/80 (35%) of patients. At multivariate analysis, absence of tumoral PD-L1 staining [odd ratio (OR) 10.8, 95% confidence interval (CI) 2.7-43.3, P < 0.001] and the presence of TIMC (OR 6.5, 95% CI 1.7-24.3, P < 0.005) were associated with a better response to treatment. Median progression-free survival (PFS) and overall survival were 10 and 15 months, respectively. By multivariate assessment, PD-L1 expression [hazard ratio (HR) 4.3, 95% CI 2.1-8.7, P < 0.0001] and absence of TIMC (HR 2.5, 95% CI 1.4-4.7, P < 0.002) correlated with shorter PFS. PD-L1 overexpression (HR 6.2, 95% CI 2.8-14.2, P < 0.0001) and absence of TIMC (HR 3.1, 95% CI 1.5-6.5, P < 0.002) were independent prognostic factors for melanoma-specific survival. CONCLUSION Our results provide the first proof-of-principle evidence for the predictive and prognostic relevance of PD-L1 immunohistochemical expression and density of immune cell infiltration in BRAF(V600)-mutated MMP treated with BRAFi.

The Krüppel-like factor 2 transcription factor gene is recurrently mutated in splenic marginal zone lymphoma

The Kruppel-like factor 2 transcription factor gene is recurrently mutated in splenic marginal zone lymphoma

iNOS activity is necessary for the cytotoxic and immunogenic effects of doxorubicin in human colon cancer cells

BackgroundDoxorubicin is one of the few chemotherapeutic drugs able to exert both cytotoxic and pro-immunogenic effects against cancer cells. Following the drug administration, the intracellular protein calreticulin is translocated with an unknown mechanism onto the plasma membrane, where it triggers the phagocytosis of tumour cells by dendritic cells. Moreover doxorubicin up-regulates the inducible nitric oxide (NO) synthase (iNOS) gene in cancer cells, leading to huge amounts of NO, which in turn acts as a mediator of the drug toxicity and as a chemosensitizer agent in colon cancer. Indeed by nitrating tyrosine on the multidrug resistance related protein 3, NO decreases the doxorubicin efflux from tumour cells and enhances the drug toxicity. It is not clear if NO, beside playing a role in chemosensitivity, may also play a role in doxorubicin pro-immunogenic effects. To clarify this issue, we compared the doxorubicin-sensitive human colon cancer HT29 cells with the drug-resistant HT29-dx cells and the HT29 cells silenced for iNOS (HT29 iNOS-).ResultsIn both HT29-dx and HT29 iNOS- cells, doxorubicin did not induce NO synthesis, had a lower intracellular accumulation and a lower toxicity. Moreover the drug failed to promote the translocation of calreticulin and the phagocytosis of HT29-dx and HT29 iNOS-cells, which resulted both chemoresistant and immunoresistant. However, if NO levels were exogenously increased by sodium nitroprusside, the chemosensitivity to doxorubicin was restored in HT29 iNOS-cells. In parallel the NO donor per se was sufficient to induce the exposure of calreticulin and to increase the phagocytosis of HT29 iNOS- cells by DCs and their functional maturation, thus mimicking the pro-immunogenic effects exerted by doxorubicin in the parental drug-sensitive HT29 cells.ConclusionOur data suggest that chemo- and immuno-resistance to anthracyclines are associated in colon cancer cells and rely on a common mechanism, that is the inability of doxorubicin to induce iNOS. Therefore NO donors might represent a promising strategy to restore both chemosensitivity and immunosensitivity to doxorubicin in resistant cells.

Immunogenicity of 56°C and UVC-treated prostate cancer is associated with release of HSP70 and HMGB1 from necrotic cells

Prostate hyperthermia and photodynamic therapy can be delivered by a variety of procedures which result in a wide range of temperatures and light energy and cause different kinds of cell death.