Gaia C. Ghedini

Improved Clinical Outcome in Indolent B-Cell Lymphoma Patients Vaccinated with Autologous Tumor Cells Experiencing Immunogenic Death

Increasing evidence argues that the success of an anticancer treatment may rely on immunoadjuvant side effects including the induction of immunogenic tumor cell death. Based on the assumption that this death mechanism is a similar prerequisite for the efficacy of an active immunotherapy using killed tumor cells, we examined a vaccination strategy using dendritic cells (DC) loaded with apoptotic and necrotic cell bodies derived from autologous tumors. Using this approach, clinical and immunologic responses were achieved in 6 of 18 patients with relapsed indolent non-Hodgkins lymphoma (NHL). The present report illustrates an impaired ability of the neoplastic cells used to vaccinate nonresponders to undergo immunogenic death on exposure to a cell death protocol based on heat shock, γ-ray, and UVC ray. Interestingly, when compared with doxorubicin, this treatment increased surface translocation of calreticulin and cellular release of high-mobility group box 1 and ATP in histologically distinct NHL cell lines. In contrast, treated lymphoma cells from responders displayed higher amounts of calreticulin and heat shock protein 90 (HSP90) compared with those from nonresponders and boosted the production of specific antibodies when loaded into DCs for vaccination. Accordingly, the extent of calreticulin and HSP90 surface expression in the DC antigenic cargo was significantly associated with the clinical and immunologic responses achieved. Our results indicate that a positive clinical effect is obtained when immunogenically killed autologous neoplastic cells are used for the generation of a DC-based vaccine. Therapeutic improvements may thus be accomplished by circumventing the tumor-impaired ability to undergo immunogenic death and prime the antitumor immune response.

Long pentraxin 3: A novel multifaceted player in cancer

Since its discovery in 1992, long pentraxin 3 (PTX3) has been characterized as soluble patter recognition receptor, a key player of the innate immunity arm with non-redundant functions in pathogen recognition and inflammatory responses. As a component of the extra-cellular matrix milieu, PTX3 has been implicated also in wound healing/tissue remodeling, cardiovascular diseases, fertility, and infectious diseases. Consequently, PTX3 levels in biological fluids have been proposed as a fluid-phase biomarker in different pathological conditions. In the last decade, experimental evidences have shown that PTX3 may exert a significant impact also on different aspects of cancer biology, including tumor onset, angiogenesis, metastatic dissemination and immune-modulation. However, it remains unclear whether PTX3 acts as a good cop or bad cop in cancer. In this review, we will summarize and discuss the scientific literature data focusing on the role of PTX3 in experimental and human tumors, including its putative translational implications.

Future applications of FGF/FGFR inhibitors in cancer

ABSTRACT Introduction: Deregulation of the fibroblast growth factor (FGF)/FGF receptor (FGFR) network occurs frequently in tumors due to gene amplification, activating mutations, and oncogenic fusions. Thus, the development of FGF/FGFR-targeting therapies is the focus of several basic, preclinical, and clinical studies. Areas covered: This review will recapitulate the status of current FGF/FGFR-targeted drugs. Expert commentary: Non-selective FGF/FGFR inhibitors have been approved for cancer treatment but evidence highlights various complications affecting their use in the clinical practice. It appears mandatory to identify FGF/FGFR alterations and appropriate biomarkers that may predict and monitor response to treatment, to establish the contribution of the FGF/FGFR system to the onset of mechanisms of drug resistance, and to develop effective combinations of FGF/FGFR inhibitors with other targeted therapies.

Abstract 2637: Role of d16HER2 splice variant in HER2-positive breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Almost 90% of human primary breast cancers (BCs) express 4-9% of total wild-type (WT) HER2 as a splice variant lacking exon 16 (d16HER2). Consequent in-frame activating deletion of 2 cysteine residues causes an imbalanced conformation resulting in receptor constitutive homodimerization, enhanced signaling activity, transformation and altered trastuzumab (T) binding. We recently produced a human d16HER2 transgenic (tg) mouse characterized by a rapid multifocal mammary tumors onset and the expression of active d16HER2 dimers on tumor cells, whose downstream signaling was found coupled to multiple activated nodes that include Src kinase. In order to dissect d16HER2 role in the aggressiveness and in the susceptibility to anti-HER2 therapy, we focused on the generation of stable d16HER2-expressing mammary tumor cell lines to be used in different bioassays. An immunomagnetic purification procedure was applied to generate primary homogeneously d16HER2-expressing cell cultures. These purified tumor cell lines were analyzed by flow cytometry and immunofluorescence and their migration/invasion ability was assessed through Boyden chamber test. d16HER2 downstream signaling was evaluated by western blot and T, Lapatinib (L) and Dasatinib (D) drugs sensitivity was measured with WST-1 and soft-agar bioassays. As controls, we compared in vitro d16HER2-models oncogenic features to those of the human BC BT474, which also co-expresses a low amount of d16HER2 transcript (4%), and to a murine mammary carcinoma cell line (wtHER2), derived from a primary mammary tumor developed in human WT HER2 tg mice. We found that d16HER2 in vitro models expressed high levels of stable homodimers combined with the recruitment of activated Src, STAT3, MAPK and Akt, as in vivo primary mammary tumors. Both in bidimensional (2D) and matrigel-cultured tumor cells, we confirmed the T lower binding capability for d16HER2 than other anti-HER2 MAbs directed to different extracellular domain epitopes. d16HER2 tumor cells had an enhanced migratory and invasive capacity compared to wtHER2 and BT474 cells and, notably, were completely resistant to T treatment and less responsive to L. In virtue of a highly activated Src kinase expression in d16HER2-positive models, we tested in 2D the therapeutic effects of D and observed that d16HER2-cells were significantly more sensitive than wtHER2 and BT474 cells. Preliminary 3D-drug susceptibility assays showed that the sensitivity of d16HER2 cells increased for all the tested drugs, if assessed in a suitable environment such as soft-agar. Our findings further indicate that the constitutive expression of d16HER2 variant identifies an aggressive tumor phenotype and confirm, at least in vitro in 2D conditions, that this isoform is resistant to T and L, whereas is sensitive to D. Further analyses are ongoing to analyze in vivo drug sensitivity of d16 in comparison to WT HER2 model. Supported by AIRC and Ministry of Health Citation Format: Gaia C. Ghedini, Arianna Palladini, Valentina Ciravolo, Lorenzo Castagnoli, Giulia Marzano, Roberta Zappasodi, Guido Santilli, Augusto Amici, Alessia Lamolinara, Manuela Iezzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. Role of d16HER2 splice variant in HER2-positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2014-2637

Abstract 916: Role of delta16HER2 splice variant in HER2-driven tumor progression and response to targeted therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We reported that the splice variant of human HER2 lacking exon 16 (delta16HER2) represents a highly penetrating HER2 oncogenic alteration identified in human primary breast tumor specimens and is able to influence the response to Trastuzumab. This HER2 variant forms covalent cysteine bonds that generate constitutively active homodimers, thereby activating multiple oncogenic downstream signaling pathways that we recently found to be mediated through activated Src kinase. To examine the ability of delta16HER2 to transform mammary epithelium in vivo and to monitor delta16HER2-driven tumorigenesis in live mice, we generated a FVB transgenic mouse model for the human delta16HER2 isoform. Transgenic female mice developed multifocal mammary tumors with a rapid onset starting at about 12 weeks of age and progressively thereafter, clearly pointing to the candidacy of the delta16HER2 isoform as the transforming form of the human HER2 oncoprotein. Histological and immunohistochemical analysis (IHC) of primary mammary nodules revealed a population of polygonal cells with classical epithelia-like aspects distinctly expressing HER2 and also a population of smaller spindle-shaped cells arranged in fascicles with lower levels of HER2 expression, suggesting the onset of the epithelial-to-mesenchymal transition (EMT). Consistent with these findings, FACS analysis of delta16HER2-positive tumor cells immunomagnetically purified from disaggregated transgenic primary tumors indicated the increased mean fluorescence intensity of HER2 staining with increasing tumor cell size. IHC analysis of the lung metastases that had formed in the majority of female mice revealed monomorphic and classical epithelial tumor cells homogeneously expressing high levels of delta16HER2. FACS and IHC analyses confirmed the lower binding efficacy of Trastuzumab to delta16HER2-overexpressing primary tumor cells cultured both under bidimensional (2D) and tridimensional (3D) conditions as compared to monoclonal reagents directed to different HER2 extracellular domain epitopes. Experiments in both primary and metastatic in vitro and in vivo delta16HER2-positive models are in progress to determine whether delta16HER2-driven tumor aggressiveness and Trastuzumab susceptibility depend not only on genetic changes intrinsic to the tumor cell, i.e., the EMT process, but also on extrinsic tumor surrounding microenvironment-related factors such as an imbalance between extracellular and intracellular pH, redox state and hypoxia. Preliminary FACS and IHC analyses indicate that delta16HER2-positive primary tumor cells are reactive for known epithelial markers as EpCAM, E-cadherin- and ck-18 and, a small subset of these mammary tumor cells, also stain positive for the mesenchymal differentiation markers vimentin, N-cadherin and ck14 significantly indicating an active EMT program. Supported by AIRC and Ministry of Health Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916. doi:1538-7445.AM2012-916

Abstract 4785: Identification of HSP105 as a novel non-Hodgkin lymphoma (NHL) restricted antigen

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We recently reported that vaccination using dendritic cells (DCs) loaded with killed autologous tumor cells in relapsed indolent NHL patients induced objective clinical responses that correlate with multifaceted immunological responses. In particular, since responders (R) showed tumor-restricted humoral immunity in autologous setting, we exploited antibody (Ab) repertoires from vaccinated patients to evaluate whether the observed antibody responses was directed to indolent NHL-shared antigens (ags). Immunohistochemistry and flow-cytometric analyses of biotinylated Immunoglobulins (Igs) from vaccinated patients’ serum samples preadsorbed on normal isolated B cells in the attempt to decrease Abs cross-reacting with normal auto-ags showed that only post-vaccine serum Ig of responders specifically reacted with higher intensity on allogeneic NHL tumors (either long term cell cultures or primary tumors from the same series) compared to matched biotinylated Igs from control samples (pre-vaccine serum from responders, pre- and post-vaccine serum from non-responders and healthy donors). In addition, only Abs contained in post-vaccine serum from R significantly inhibited NHL DOHH2 cells growth strongly suggesting the presence of therapeutic Abs in their serum samples. These findings indicate that Igs from R recognize tumor cell-surface associated molecules different from patient-specific idiotypes and support the potential expression of novel NHL-restricted ags. As a step toward identifying NHL-restricted ags as potential novel therapeutic targets, we applied a properly modified serological proteome analysis (SERPA) using as target DOHH2 cells and as probes pre- and post-vaccine Ab repertoires from our series. Proteins, fractionated using an OFFGEL IPG strip (pH 3-10), were separated on SDS-PAGE and then immunoblotted with pre- and post-vaccine Igs from R and controls. Differential hybridization analyses have revealed some potential candidate tumor-associated antigens which include heat shock protein 105 (HSP105), whose immunological role has been already proved in solid tumors. Preliminary data indicate that HSP105 is also expressed on cell membrane from different tumor B cell lines and experiments are in progress to evaluate its potential implication in tumor growth control. Partially supported by AIRC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4785.