A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies

INTRODUCTION: The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy. METHODS: Blood samples were collected from 61 locally advanced breast cancers (36 HER2- and 25 HER2+) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8+ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data. RESULTS: The proportion of circulating immune effectors was similar in HER2+ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4+/CD8+ T cell ratio (with a prevalence of naïve and central memory CD8+ T cells) were observed in HER2- cases. Higher numbers of circulating CD8+ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2+ cases, together with a higher prevalence of intratumor CD8+ T cells. Serum cytokine profile of HER2+ patients was similar to that of controls, whereas HER2- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2+ cases (IL-2, IL-1β, IL-8, IL-6, IL-10). CONCLUSIONS: Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects

Retinoic acid and α-Interferon combination as therapy for Akt-driven non-Hodgkin lymphomas

Given the critical role of the PI3K/Akt pathway in cell growth and survival, it is not surprising that constitutive activation of this pathway contributes to the pathogenesis of many types of lymphoid malignancies, including mantle cell lymphoma (MCL), follicular lymphoma (FL), and cutaneous T-cell lymphoma (CTCL). Available drugs aimed to strike the PI3K/Akt pathway in these tumors are unfortunately dampened by relevant toxicities. Therefore, more effective and safer therapeutic options targeting Akt are needed. Herein we demonstrated that the combination of 9-cis-retinoic acid (RA) and Interferon-alpha (IFN-\u3b1) induces marked anti-proliferative and pro-apoptotic effects in MCL cells through the modulation of critical targets. Particularly, IFN-\u3b1 enhances 9-cis-RA-mediated G0/G1 cell accumulation by down-regulating cyclin D1 and increasing p27Kip1 and p21WAF1/Cip1 protein levels. Furthermore, 9-cis-RA/IFN-\u3b1 combination induces MCL apoptosis by triggering both caspase 8 and caspase 9 resulting in Bax and Bak activation, and up-regulating the pro-apoptotic proteins Noxa and PLSCR1. In particular, sequestration of the anti-apoptotic proteins Mcl-1 and A1/Bfl1 by up-regulated Noxa results in the activation of Bid, and the consequent induction of apoptosis is inhibited by Noxa silencing. PLSCR1silencing demonstrated a role for this protein not only in 9-cis-RA/IFN-\u3b1-induced apoptosis but also in 9-cis-RA/IFN-\u3b1-dependent sensitization to anti-tumor agents currently used in the clinical practice for MCL management, such as doxorubicin and bortezomib. These drugs are able to further increase PLSCR1 expression in 9-cis-RA/IFN-\u3b1 pre-treated MCL cells. Moreover, immunohistochemical analysis of MCL tumor biopsies and primary cultures revealed a variable expression of endogenous PLSCR1 in this setting, an heterogeneity that stimulates the search of possible correlations with clinical-pathological parameters, particularly with those related to the response to pro-apoptotic drugs. In future perspective, in fact, analysis of PLSCR1 expression might allow the identification of tumors more prone to undergo apoptosis, and strategies able to up-regulate PLSCR1, like 9-cis-RA/IFN-\u3b1 combination, might successfully complement and improve conventional treatment modalities. Notably, we also found that 9-cis-RA/IFN-\u3b1-induced PLSCR1 up-regulation occurs through STAT1 activation in dependence on Akt pathway. In addition, we demonstrated that 9-cis-RA/IFN-\u3b1 co-treatment is able to trigger apoptotic effects also in FL and CTCL cells, and, more interesting, the 9-cis-RA/IFN-\u3b1-dependent apoptosis is associated with the inhibition of Akt constitutive activation in the different NHL histotypes analyzed. In particular, Noxa up-regulation in MCL, FL, and CTCL lymphoma cells is associated with nuclear translocation of the FOXO3a transcription factor as a consequence of the 9-cis-RA/IFN-\u3b1-induced Akt but not of mTOR inhibition. Indeed, pharmacological suppression of Akt, but not of TORC1, induces apoptosis in FL cell lines, and increases Noxa protein levels in MCL cells, supporting the conclusion that inhibition of the Akt pathway, the resulting FOXO3a activation, and Noxa up-regulation are the critical molecular mechanisms underlying 9-cis-RA/IFN-\u3b1-induced cell death in different type of lymphoid malignancies. These results strengthen the role of Akt as a clinically relevant molecular target and support the potential therapeutic value of RA/IFN-\u3b1 combination to improve the management of Akt-driven non-Hodgking lymphoma

Epstein-Barr virus and telomerase: from cell immortalization to therapy

Overcoming cellular senescence is strictly required for virus-driven tumors, including those associated with Epstein-Barr virus (EBV). This critical step is successfully accomplished by EBV through TERT expression and telomerase activation in infected cells. We herein review the complex interplay between EBV and TERT/telomerase in EBV-driven tumorigenesis. Evidence accumulated so far clearly indicates that elucidation of this issue may offer promising opportunities for the design of innovative treatment modalities for EBV-associated malignancies. Indeed, several therapeutic strategies for telomerase inhibition have been developed and are being investigated in clinical trials. In this respect, our recent finding that TERT inhibition sensitizes EBV+ lymphoma cells to antivirals through activation of EBV lytic replication is particularly promising and provides a rationale for the activation of clinical studies aimed at assessing the effects of combination therapies with TERT inhibitors and antivirals for the treatment of EBV-associated malignancies

Preliminary results from the first geomagnetic deep sounding in the Western sector of the Anti Atlas region, Southern Morocco

An array of vector magnetometers was temporarily installed in the Western sector of the Anti Atlas chain, Morocco in the frame of the Geomagnetic Depth Sounding (GDS) technique. As a fruitful collaboration between Italy and Morocco, the joint project “Terremagnet”, funded by the Italian Ministry of the Foreign Affair, was aimed to local observations of the Earth’s magnetic field in order to define electric conductivity horizontal and vertical contrasts in a tectonic active region. The analysis in time and frequency-domain for tests on the induced EM field dimension, computations of single site and coupled site Transfer Functions (TFs) and induction vectors configuration are shown. Recorded data are compared to Averroes geomagnetic observatory (Morocco) (IAGA code: AVE, lat. 33° 17’ 53” N, long. 7° 24’ 48” W, altitude: 230 m a.s.l.). The preliminary results from Morocco are really encouraging and pointed out good data samplings that will allow shedding up light on the tectonic setting of this peculiar region of the Anti-Atlas

hTERT inhibition triggers Epstein-Barr virus lytic cycle and apoptosis in immortalized and transformed B cells: a basis for new therapies

Purpose: Induction of viral lytic cycle, which induces death of host cells, may constitute a useful adjunct to current therapeutic regimens for Epstein-Barr virus (EBV)-driven malignancies. Human telomerase reverse transcriptase (hTERT), essential for the oncogenic process, may modulate the switch from latent to lytic infection. The possible therapeutic role of hTERT inhibition combined with antiviral drugs was investigated. Experimental Design: EBV-negative BL41 and convertant EBV-positive BL41/B95.8 Burkitt's lymphoma cell lines and lymphoblastoid cell lines (LCL) were infected with retroviral vector encoding short hairpin RNA (shRNA) anti-hTERT and cultured with or without the prodrug ganciclovir. The effects on EBV lytic replication, cell proliferation, and apoptosis were characterized. Results: hTERT silencing by shRNA induced the expression of BZLF1, EA-D, and gp350 EBV lytic proteins and triggered a complete lytic cycle. This effect was associated with downregulation of BATF, a negative regulator of BZLF1 transcription. hTERT silencing also resulted in antiproliferative and proapoptotic effects. In particular, hTERT inhibition induced an accumulation of cells in the S-phase, an effect likely due to the dephosphorylation of 4E-BP1, an AKT1-dependent substrate, which results in a decreased availability of proteins needed for cell-cycle progression. Besides inducing cell death through activation of complete EBV lytic replication, hTERT inhibition triggered AKT1/FOXO3/NOXA-dependent apoptosis in EBV-positive and -negative Burkitt's lymphoma cells. Finally, ganciclovir enhanced the apoptotic effect induced by hTERT inhibition in EBV-positive Burkitt's lymphomas and LCLs. Conclusions: These results suggest that combination of antiviral drugs with strategies able to inhibit hTERT expression may result in therapeutically relevant effects in patients with EBV-related malignancies

Broadening specificity and enhancing cytotoxicity of adoptive T cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxorubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-\u3baB activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable

Lymphomagenic properties of a HIV p17 variant derived from a splenic marginal zone lymphoma occurred in a HIV-infected patient

Despite antiretroviral therapy, HIV+ individuals still have increased risk to develop lymphomas, including marginal zone lymphomas, suggesting that factors other than HIV-related immunosuppression are probably acting as lymphomagenic factors in the HIV setting. The possible pathogenic involvement of HIV p17 protein variants was investigated in a particularly informative case of HIV-related splenic marginal zone lymphoma, which was negative for oncogenic virus infections, thus allowing us to assess the possible direct contribution of these HIV-encoded proteins to lymphomagenesis. The presence of p17 protein was analyzed by immunohistochemistry in lymphoma tissue. Recombinant p17 protein derived from the dominant sequence detected in plasma and lymphoma biopsy was characterized for B-cell proliferation, clonogenicity in soft agar, in vitro tube formation and wound healing. Intracellular signaling was investigated by immunoblotting. HIV p17 protein was detected in reactive lymphoid follicles but not within lymphoma cells. An identical dominant variant p17 sequence, p17-Lyrm, carrying a 117 to 118 Ala-Ala insertion was detected in both plasma and lymphoma tissue. Recombinant p17-Lyrm enhanced B-cell proliferation and clonogenicity promoted the formation of capillary-like structures and enhanced endothelial cell migration. Unlike reference p17, the p17-Lyrm variant enhanced the activation of Akt and ERK, critical kinases in lymphomagenesis. p17-Lyrm clonogenic activity was dependent on the activation of Akt but not of ERK1/2. These results indicated that HIV p17 variants with distinct molecular signatures and functional properties may accumulate in lymphoid tissues of HIV-infected individuals where they may act as a local stimulus promoting the development of lymphomas

Broadening specificity and enhancing cytotoxicity of adoptive T Cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxo-rubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-kappa B activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable