Angela Vasaturo

Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses.

ABSTRACT Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here, we compared the efficacy of the invariant NKT (iNKT) cell agonist α-galactosylceramide (α-GalCer) and TLR ligands (R848 and poly I:C) as an adjuvant for the full length ovalbumin (OVA) in PLGA nanoparticles. We observed that OVA+α-GalCer nanoparticles (NP) are superior over OVA+TLR-L NP in generating and stimulating antigen-specific cytotoxic T lymphocytes without the need for CD4+ T cell help. Not only a 4-fold higher induction of antigen-specific T cells was observed, but also a more profound IFN-γ secretion was obtained by the addition α-GalCer. Surprisingly, we observed that mixtures of OVA containing NP with α-GalCer were ineffective, demonstrating that co-encapsulation of both α-GalCer and antigen within the same nanoparticle is essential for the observed T cell responses. Moreover, a single immunization with OVA+α-GalCer NP provided substantial protection from tumor formation and even delayed the growth of already established tumors, which coincided with a prominent and enhanced antigen-specific CD8+ T cell infiltration. The provided evidence on the advantage of antigen and α-GalCer coencapsulation should be considered in the design of future nanoparticle vaccines for therapeutic purposes.

T-cell Landscape in a Primary Melanoma Predicts the Survival of Patients with Metastatic Disease after Their Treatment with Dendritic Cell Vaccines

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3(+) T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496-506. ©2016 AACR.

Semaphorin 7A Promotes Chemokine-Driven Dendritic Cell Migration

Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.

Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20+ cells in B cell follicles in human spleen and appendix. CD3+ T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

Multispectral imaging for highly accurate analysis of tumour-infiltrating lymphocytes in primary melanoma

The quality and quantity of the infiltration of immune cells into tumour tissues have substantial impacts on patients’ clinical outcomes, and are associated with response to immunotherapy. Therefore, the precise analysis of tumour‐infiltrating lymphocytes (TILs) is becoming an important additional pathological biomarker. Analysis of TILs is usually performed semiquantitatively by pathologists on haematoxylin and eosin‐stained or immunostained tissue sections. However, automated quantification outperforms semiquantitative approaches, and is becoming the standard. Owing to the presence of melanin pigment, this approach is seriously hampered in melanoma, because the spectrum of melanin lies close to that of commonly used immunohistochemical stains. Aim of this study is to overcome the technical issues due to the presence of melanin for an automated and accurate quantification of TILs in melanoma.

Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity

Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n = 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n = 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.

Immune infiltrates impact on the prediction of prognosis and response to immunotherapy of melanoma patients

Background Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite specific therapies have been explored for many years, no effective therapeutic options have been developed. Vaccination strategies, including Dendritic Cells (DC) based immunotherapy, are consistently increasing the proportion of cancer patients with anti-vaccine immune responses although the number of patients with increased overall survival is still limited. The efficacy of the immunotherapy is mainly dependent on tumor microenvironment –immune system interactions. Our aim is to evaluate the host immune response to melanoma by quantifying the density and location of T cells in primary tumors of patients treated with DC immunotherapy and correlate them with clinical variables such as overall survival (OS). Materials and methods We collected 60 FFPE primary tumors from melanoma patients treated with DC immunotherapy. Serial sections (4 micron in thickness) were stained with CD3, CD8 and CD45RO antibodies. Haematoxylin was used as a counterstain and Nova Red for the immunohistochemical stain. All the slides were digitalized and an automated quantitative analysis was performed in order to evaluate the density and location of two lymphocyte populations, cytotoxic (CD8) and memory (CD45RO) T cells. Of all samples the clinical outcome of the patient is known. Results The immunohistochemical analysis of primary melanoma using a small set of patients resulted in significant differences between short (OS 24 months). A high degree of T cells infiltration was seen in the tumor area of both patients, responding and non-responding to DC immunotherapy. However, the location but not the density of TILs was significantly different in the two cohorts of patients and showed a strong correlation with clinical response to DC vaccination. Conclusions Immune cells within melanoma tumors may have a prognostic value and clinical significance as a predictor of patient outcome and response to DC immunotherapy in melanoma patients.

Prognostic value of intra-tumoral CD8+/FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis

Patients with isolated colorectal‐cancer‐liver‐metastases (CRCLM) frequently undergo metastatectomy. Tumor‐infiltrating‐lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor‐infiltrating CD8+ cytotoxic T‐cells and FoxP3+ regulatory T‐cells at the metastatic site of CRCLM patients.

Abstract A016: T cell landscape within primary melanoma as a predictive biomarker of survival after cancer vaccination in patients with metastatic disease

Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite different types of immunotherapy became available for the treatment of melanoma, including adoptive T cell transfer, immune checkpoint blockade, and vaccines such as dendritic cell vaccines, when applied to treat metastatic melanoma, only 10 to 40% of the patients have long term benefit. Furthermore, given the toxicity and high costs associated with immunotherapy there is a stringent need to identify biomarkers that may predict its potential efficacy. We have investigated whether the presence and distribution of T cells within the primary tumor of melanoma patients correlates with survival when treated for metastatic disease with dendritic cell based cancer vaccines. Multispectral imaging and quantitative immunohistochemistry have been used to compare the tumor microenvironment of responding patients, that survived longer than 20 months after immunotherapy, and non-responding patients that survived less than 12 months after immunotherapy. The density and location of T cell were initially assessed in primary melanomas of 19 metastatic melanoma patients treated with dendritic cell based immunotherapy (discovery set) and subsequently in an independent cohort of 39 patients (validation cohort). In the discovery cohort we observed a very high correlation between the ratios of peritumoral over intratumoral T cells in the primary tumor and overall survival; lower peri/intratumoral T cell ratios in primary tumors were associated with improved clinical outcome indicating that the intratumoral T cell component is more prominent in responding patients whereas T cells fail to infiltrate the tumor in non-responding patients. ROC curve and multivariate analysis were exploited to evaluate the predictive power of the T cell ratio compared and/or combined to established prognostic markers. The same statistical analyses in the validation cohort confirmed our findings showing that the peri/intratumoral T cell ratio correctly predicted long term survival after DC vaccination in 90% of the cases and was the strongest predictor of survival in the multivariate analysis. This study indicates that the ratio between CD3 positive peri/intra-tumoral T cells is a very strong predictor of clinical outcome and response to dendritic cells immunotherapy in melanoma patients. Citation Format: Angela Vasaturo, Altuna Halilovic, Kalijn Bol, Dagmar Verweij, Patricia Groenen, Han van Krieken, Willeke Blokx, Jolanda de Vries, Johannes Textor, Carl G. Figdor. T cell landscape within primary melanoma as a predictive biomarker of survival after cancer vaccination in patients with metastatic disease. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A016.

T cell landscape within primary melanoma as a biomarker of survival after cancer vaccination in patients with metastatic disease

Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite different types of immunotherapy became available for the treatment of melanoma, including adoptive T cell transfer, immune checkpoint blockade, and vaccines such as dendritic cell vaccines, when applied to treat metastatic melanoma, only 10 to 40% of the patients have long term benefit. Furthermore, given the toxicity and high costs associated with immunotherapy there is a stringent need to identify biomarkers that may predict its potential efficacy. We have investigated whether the presence and distribution of T cells within the primary tumor of melanoma patients correlates with survival when treated for metastatic disease with dendritic cell based cancer vaccines. Quantitative multispectral imaging has been used to compare the tumor microenvironment of responding patients that survived longer than 20 months after immunotherapy, and non-responding patients that survived less than 12 months after immunotherapy. T cell infiltrates have been initially assessed in primary melanomas of 19 metastatic melanoma patients treated with dendritic cell based immunotherapy (discovery set) and subsequently in an independent cohort of 39 patients (validation cohort). In the discovery cohort we observed a very high correlation between the ratio of peritumoral over intratumoral T cells in the primary tumor and overall survival. Lower peri/intratumoral T cell ratios in primary tumors were associated with improved clinical outcome. ROC curve and multivariate analysis were exploited to evaluate the predictive power of the T cell ratio and established prognostic markers. Statistical analyses in the validation cohort confirmed our findings showing that the peri/intratumoral T cell ratio correctly predicted long term survival after DC vaccination in 90% of the cases and was the strongest predictor of survival in the multivariate analysis. This study indicates that the ratio between CD3 positive peri/intra-tumoral T cells is a very strong predictor of patient outcome and response to dendritic cells immunotherapy in melanoma patients.