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BACKGROUND: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma. METHODS: 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting. RESULTS: The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan-Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints. CONCLUSIONS: The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research.
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Detección Precoz del Cáncer , Melanoma , Humanos , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf , Genómica , ItaliaRESUMEN
BACKGROUND AIMS: Dendritic cells (DCs) are professional antigen-presenting cells of the mammalian immune system. Ex vivo differentiated DCs represent a unique Advanced Therapy Medicinal Product (ATMP), used in several clinical trials as personalized cancer immunotherapy. The therapy's reliability depends on its capacity to produce high-quality mature DCs (mDCs) in compliance with Good Manufacturing Practices. AIMS: From March 2010 to December 2023, 103 patients were enrolled in multiple clinical trials at the Immuno-Gene Therapy Factory at IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori". Six hundred forty-two doses were produced, and the manufacturing process was implemented to optimize production. Our study is a retrospective analysis focusing on the quality control results. METHODS: We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating viability, purity and phenotype of mDCs and their quality in terms of microbiological safety. The data obtained are given with median and interquartile range. RESULTS: The batches were found to be microbiologically safe in terms of sterility, mycoplasma, and endotoxins. An increase in DC maturation markers was found. The release criteria checks showed a high percentage of viability and purity was maintained during the production process. CONCLUSIONS: Our findings have confirmed that the measures implemented have ensured the safety of the products and have contributed to the establishing a robust "Pharmaceutical Quality System." This has enabled many safe mDCs to be produced for clinical trials.
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PURPOSE: The aims of our retrospective study investigated the role of immune system in glioblastoma (GBM), which is the most aggressive primary brain tumor in adults characterized by a poor prognosis. The recurrence rate remains high, probably due to "immune-desert" tumor microenvironment (TME) making GBM hidden from the anti-tumoral immune clearance. Considering this, we aimed to create a panel of prognostic markers from blood and tumor tissue correlating with overall survival (OS) and progression-free survival (PFS). METHODS: Firstly, we analyzed the inflammatory markers NLR and PLR as the ratio of the absolute neutrophil count and absolute platelet count by the absolute lymphocyte count respectively, collected at different time points in the peripheral blood of 95 patients. Furthermore, in 31 patients of the same cohort, we analyzed the formalin-fixed paraffin embedded samples to further compare the impact of circulating and inflammatory markers within the TME. RESULTS: Patients aged < 60 years and with methylated MGMT showed better OS. While, pre-chemotherapy Systemic Inflammatory Index (SII) < 480 was related to a better OS and PFS, we observed that only CD68+macrophage and CD66b+neutrophils expressed in vascular/perivascular area (V) showed a statistically significant prognostic role in median OS and PFS. CONCLUSIONS: Thus, we underscored a role of SII as predictive value of response to STUPP protocol. Regarding the TME-related markers, we suggested to take into consideration for future studies with new immunotherapy combinations, each component relating to expression of immune infiltrating subsets.
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Neoplasias Encefálicas , Glioblastoma , Neurocirugia , Adulto , Humanos , Glioblastoma/metabolismo , Estudios Retrospectivos , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/cirugía , Pronóstico , Neutrófilos , Linfocitos , Microambiente TumoralRESUMEN
BACKGROUND: Dendritic cells (DCs) are the most efficient antigen-presenting cells and act at the center of the immune system owing to their ability to control both immune tolerance and immunity. In cancer immunotherapy, DCs play a key role in the regulation of the immune response against tumors and can be generated ex vivo with different cytokine cocktails. METHODS: We evaluated the feasibility of dinoprostone (PGE2) replacement with the molecular analog sulprostone, in our good manufacturing practice (GMP) protocol for the generation of DC-based cancer vaccine. We characterized the phenotype and the function of DCs matured in the presence of sulprostone as a potential substitute of dinoprostone in the pro-inflammatory maturation cocktail consisting of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and IL-6. RESULTS: We found that sulprostone invariably reduces the recovery, but does not significantly modify the viability and the purity of DCs. The presence of sulprostone in the maturation cocktail increases the adhesion of single cells and of clusters of DCs to the flask, making them more similar to their immature counterpart in terms of adhesion and spreading proprieties. Moreover, we observed that sulprostone impairs the expression of co-stimulatory molecules and the spontaneous as well as the directed migration capacity of DCs. DISCUSSION: These findings underscore that the synthetic analog sulprostone strongly reduces the functional quality of DCs, thus cannot replace dinoprostone in the maturation cocktail of monocyte-derived DCs.
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Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Dinoprostona/análogos & derivados , Dinoprostona/farmacología , Vacunas contra el Cáncer/inmunología , Células Cultivadas , Células Dendríticas/fisiología , Evaluación Preclínica de Medicamentos , Humanos , Inmunoterapia Adoptiva/métodos , Interleucina-1beta/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/fisiología , Equivalencia Terapéutica , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Lenalidomide is an immunomodulatory agent clinically active in chronic lymphocytic leukemia patients. The specific mechanism of action is still undefined, but includes modulation of the microenvironment. In chronic lymphocytic leukemia patients, nurse-like cells differentiate from CD14(+) mononuclear cells and protect chronic lymphocytic leukemia cells from apoptosis. Nurse-like cells resemble M2 macrophages with potent immunosuppressive functions. Here, we examined the effect of lenalidomide on the monocyte/macrophage population in chronic lymphocytic leukemia patients. We found that lenalidomide induces high actin polymerization on CD14(+) monocytes through activation of small GTPases, RhoA, Rac1 and Rap1 that correlated with increased adhesion and impaired monocyte migration in response to CCL2, CCL3 and CXCL12. We observed that lenalidomide increases the number of nurse-like cells that lost the ability to nurture chronic lymphocytic leukemia cells, acquired properties of phagocytosis and promoted T-cell proliferation. Gene expression signature, induced by lenalidomide in nurse-like cells, indicated a reduction of pivotal pro-survival signals for chronic lymphocytic leukemia, such as CCL2, IGF1, CXCL12, HGF1, and supported a modulation towards M1 phenotype with high IL2 and low IL10, IL8 and CD163. Our data provide new insights into the mechanism of action of lenalidomide that mediates a pro-inflammatory switch of nurse-like cells affecting the protective microenvironment generated by chronic lymphocytic leukemia into tissues.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Talidomida/análogos & derivados , Microambiente Tumoral/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Western Blotting , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Lenalidomida , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Activación de Linfocitos , Macrófagos/metabolismo , Macrófagos/patología , Monocitos/metabolismo , Monocitos/patología , Talidomida/farmacología , Células Tumorales CultivadasRESUMEN
No prospective data were available prior to 2021 to inform selection between combination BRAF and MEK inhibition versus dual blockade of programmed cell death protein-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) as first-line treatment options for BRAFV600-mutant melanoma. SECOMBIT (NCT02631447) was a randomized, three-arm, noncomparative phase II trial in which patients were randomized to one of two sequences with immunotherapy or targeted therapy first, with a third arm in which an 8-week induction course of targeted therapy followed by a planned switch to immunotherapy was the first treatment. BRAF/MEK inhibitors were encorafenib plus binimetinib and checkpoint inhibitors ipilimumab plus nivolumab. Primary outcome of overall survival was previously reported, demonstrating improved survival with immunotherapy administered until progression and followed by BRAF/MEK inhibition. Here we report 4-year survival outcomes, confirming long-term benefit with first-line immunotherapy. We also describe preliminary results of predefined biomarkers analyses that identify a trend toward improved 4-year overall survival and total progression-free survival in patients with loss-of-function mutations affecting JAK or low baseline levels of serum interferon gamma (IFNy). These long-term survival outcomes confirm immunotherapy as the preferred first-line treatment approach for most patients with BRAFV600-mutant metastatic melanoma, and the biomarker analyses are hypothesis-generating for future investigations of predictors of durable benefit with dual checkpoint blockade and targeted therapy.
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Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Ipilimumab/uso terapéutico , Inmunoterapia/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Neoplasias Cutáneas/genética , MutaciónRESUMEN
Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792 ± 86 cells/µL versus 485 ± 46 cells/µL, P=0.003). Higher numbers of non-classical CD14(+)CD16(++) and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated "education" of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.
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Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Monocitos/patología , Fagocitosis/genética , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Leucemia Linfocítica Crónica de Células B/patología , Prueba de Cultivo Mixto de Linfocitos/métodos , Masculino , Persona de Mediana Edad , Monocitos/metabolismoRESUMEN
Deletion on the long arm of chromosome 11 occurs in 5-20% of chronic lymphocytic leukaemia (CLL) patients. We analysed clinical-biological characteristics of 131 CLL patients carrying 11q deletion documented before therapy (de novo 11q deleted CLL). De novo 11q deleted CLL were characterized by high frequencies of unmutated immunoglobulin variable heavy genes, multiple fluorescence in situ hybridization aberrations and lymph node involvement. Factors significantly associated with shorter time to first treatment (TTFT) were advanced Binet stages, high white blood cell count, increased ß2 -microglobulin levels, 17p in addition, splenomegaly and more extensive lymphadenopathy. We found that patients with <25% 11q deleted nuclei (n = 22) experienced longer TTFT compared with patients with ≥25% 11q deleted nuclei (n = 87; median TTFT, 40 vs. 14 months, p = 0.011) and also showed better response to treatments (complete response, 50% vs. 21%, p = 0.016). The variables identified by multivariate analysis as independently associated with reduced TTFT were advanced Binet stages [hazard ratio (HR) 4.69; p < 0.001] and ≥25% 11q deleted nuclei (HR 4.73; p = 0.004). De novo 11q deleted CLLs exhibit variable clinical outcome. The percentage of deleted nuclei inside leukemic clone should be included in the prognostic definition of therapy-naïve 11q deleted CLL patients.
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Deleción Cromosómica , Cromosomas Humanos Par 11 , Leucemia Linfocítica Crónica de Células B/genética , Adulto , Anciano , Anciano de 80 o más Años , Heterogeneidad Genética , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/terapia , Persona de Mediana Edad , Pronóstico , Eliminación de SecuenciaRESUMEN
Cancer immunotherapy embraces many current, promising therapeutic approaches to eradicate tumors by activating host antitumor activity [...].
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BACKGROUND: Chronic lymphocytic leukemia B cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interactions with endothelial cells in infiltrated tissues and during recirculation may have a pathogenic role in chronic lymphocytic leukemia. DESIGN AND METHODS: We evaluated apoptosis of leukemic cells after co-culture on a monolayer of human umbilical vein endothelial cells with addition of fludarabine and antibodies that block adhesion. Then, we compared microarray-based gene expression profiles between leukemic cells at baseline and after co-culture. RESULTS: We found that the endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days of co-culture. Moreover, the endothelial layer decreased the sensitivity of chronic lymphocytic leukemia B cells to fludarabine-induced apoptosis. Physical contact with endothelium mediated by both ß(1)- and ß(2)- integrins is essential for the survival advantage of leukemic cells. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B cells led to the almost complete abrogation of the survival advantage (>70% inhibition of viability). However, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes in chronic lymphocytic leukemia B cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, an increase of genes involved in TGFß and Wnt signaling pathways, secretion of cytokines recruiting stromal cells and macrophages and up-regulation of anti-apoptotic molecules such as Bcl2 and Survivin. CONCLUSIONS: Our study supports the notion that endothelial cells are major players in the chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly supports survival, protects from drug-induced apoptosis and extensively modifies the gene expression profile of leukemic cells.
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Linfocitos B/metabolismo , Antígenos CD18/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Integrina beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Anticuerpos Neutralizantes/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Antígenos CD18/genética , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Comunicación Celular/efectos de los fármacos , Comunicación Celular/genética , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Integrina beta1/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Cultivo Primario de Células , Transducción de Señal/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
Advanced therapy medical products (ATMPs) are rapidly growing as innovative medicines for the treatment of several diseases. Hence, the role of quality analytical tests to ensure consistent product safety and quality has become highly relevant. Several clinical trials involving dendritic cell (DC)-based vaccines for cancer treatment are ongoing at our institute. The DC-based vaccine is prepared via CD14+ monocyte differentiation. A fresh dose of 10 million DCs is administered to the patient, while the remaining DCs are aliquoted, frozen, and stored in nitrogen vapor for subsequent treatment doses. To evaluate the maintenance of quality parameters and to establish a shelf life of frozen vaccine aliquots, a stability program was developed. Several parameters of the DC final product at 0, 6, 12, 18, and 24 months were evaluated. Our results reveal that after 24 months of storage in nitrogen vapor, the cell viability is in a range between 82% and 99%, the expression of maturation markers remains inside the criteria for batch release, the sterility tests are compliant, and the cell costimulatory capacity unchanged. Thus, the data collected demonstrate that freezing and thawing do not perturb the DC vaccine product maintaining over time its functional and quality characteristics.
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Diet is a major driver of gut microbiota variation and plays a role in metabolic disorders, including metabolic syndrome (MS). Mycorrhized foods from symbiotic agriculture (SA) exhibit improved nutritional properties, but potential benefits have never been investigated in humans. We conducted a pilot interventional study on 60 adults with ≥ 1 risk factors for MS, of whom 33 consumed SA-derived fresh foods and 27 received probiotics over 30 days, with a 15-day follow-up. Stool, urine and blood were collected over time to explore changes in gut microbiota, metabolome, and biochemical, inflammatory and immunologic parameters; previous dietary habits were investigated through a validated food-frequency questionnaire. The baseline microbiota showed alterations typical of metabolic disorders, mainly an increase in Coriobacteriaceae and a decrease in health-associated taxa, which were partly reversed after the SA-based diet. Improvements were observed in metabolome, MS presence (two out of six subjects no longer had MS) or components. Changes were more pronounced with less healthy baseline diets. Probiotics had a marginal, not entirely favorable, effect, although one out of three subjects no longer suffered from MS. These findings suggest that improved dietary patterns can modulate the host microbiota and metabolome, counteracting the risk of developing MS.
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Agricultura , Dieta , Microbioma Gastrointestinal , Síndrome Metabólico/microbiología , Adolescente , Adulto , Anciano , Dieta Saludable , Heces/microbiología , Femenino , Humanos , Italia , Masculino , Metaboloma , Persona de Mediana Edad , Proyectos Piloto , Probióticos , Adulto JovenRESUMEN
High-dose interleukin-2 (HD IL-2) has curative potential in metastatic melanoma (MM) and renal cell carcinoma (RCC). Radiotherapy (RT) kills cancer cells and induces immunomodulatory effects. Prospective trials exploring clinical and immunological properties of combined RT/HD IL-2 are still needed. We designed a phase II, single-arm clinical trial for patients with MM and RCC. The treatment schedule consisted of 3 daily doses of 6-12 Gy of RT to 1-5 non-index metastatic fields, before IL-2 at the first and third treatment cycle. HD IL-2 was administered by continuous infusion for 72 hours and repeated every 3 weeks for up to 4 cycles, thereafter every 4 weeks for a maximum of 2 cycles. The primary endpoint was the immunological efficacy of the combined RT/HD IL-2 treatment (assessed by IFN-γ ELISPOT). Nineteen out of 22 patients were evaluable for immunological and clinical response. Partial response occurred in 3 (15.7%) patients and stable disease was observed in 7 (36.8%). The disease control rate was 52.6% after a median follow up of 39.2 months. According to Common Terminology Criteria for Adverse Events 4.0 (CTCAE 4.0), the majority of toxicities were grade 1-2. Immunological responses were frequent and detected in 16 (84.2%) patients. Increased levels of IL-8 and IL-10 in melanoma, circulating effector memory CD4+ and intratumoral CD8+ T cells in both tumor types were detected after therapy. Overall the treatment was well tolerated and immunologically active. Immunomonitoring and correlative data on tumor and peripheral blood cell subsets suggest that this combination treatment could be a promising strategy for patients progressing after standard treatments.
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Antineoplásicos/administración & dosificación , Carcinoma de Células Renales/terapia , Quimioradioterapia , Interleucina-2/análogos & derivados , Neoplasias Renales/terapia , Melanoma/terapia , Neoplasias Cutáneas/terapia , Adulto , Anciano , Antineoplásicos/efectos adversos , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/secundario , Quimioradioterapia/efectos adversos , Fraccionamiento de la Dosis de Radiación , Femenino , Humanos , Infusiones Intravenosas , Interleucina-2/administración & dosificación , Interleucina-2/efectos adversos , Italia , Neoplasias Renales/inmunología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/secundario , Persona de Mediana Edad , Prueba de Estudio Conceptual , Estudios Prospectivos , Dosis de Radiación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factores de Tiempo , Resultado del TratamientoRESUMEN
Dendritic cell (DC)-based vaccination effectively induces anti-tumor immunity, although in the majority of cases this does not translate into a durable clinical response. However, DC vaccination is characterized by a robust safety profile, making this treatment a potential candidate for effective combination cancer immunotherapy. To explore this possibility, understanding changes occurring in the tumor microenvironment (TME) upon DC vaccination is required. In this line, quantitative and qualitative changes in tumor-infiltrating T lymphocytes (TILs) induced by vaccination with autologous tumor lysate/homogenate loaded DCs were investigated in a series of 16 patients with metastatic melanoma. Immunohistochemistry for CD4, CD8, Foxp3, Granzyme B (GZMB), PDL1, and HLA class I was performed in tumor biopsies collected before and after DC vaccination. The density of each marker was quantified by automated digital pathology analysis on whole slide images. Co-expression of markers defining functional phenotypes, i.e., Foxp3+ regulatory CD4+ T cells (Treg) and GZMB+ cytotoxic CD8+ T cells, was assessed with sequential immunohistochemistry. A significant increase of CD8+ TILs was found in post-vaccine biopsies of patients who were not previously treated with immune-modulating cytokines or Ipilimumab. Interestingly, along with a maintained tumoral HLA class I expression, after DC vaccination we observed a significant increase of PDL1+ tumor cells, which significantly correlated with intratumoral CD8+ T cell density. This observation might explain the lack of a significant concurrent cytotoxic reactivation of CD8+ T cell, as measured by the numbers of GZMB+ T cells. Altogether these findings indicate that DC vaccination exerts an important role in sustaining or de novo inducing a T cell inflamed TME. However, the strength of the intratumoral T cell activation detected in post-DC therapy lesions is lessened by an occurring phenomenon of adaptive immune resistance, yet the concomitant PDL1 up-regulation. Overall, this study sheds light on DC immunotherapy-induced TME changes, lending the rationale for the design of smarter immune-combination therapies.
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Linfocitos T CD8-positivos , Vacunas contra el Cáncer , Células Dendríticas , Linfocitos Infiltrantes de Tumor , Melanoma , Linfocitos T Reguladores , Vacunación , Adulto , Anciano , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Femenino , Estudios de Seguimiento , Humanos , Inflamación/inmunología , Inflamación/patología , Inflamación/terapia , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma/inmunología , Melanoma/patología , Melanoma/terapia , Persona de Mediana Edad , Metástasis de la Neoplasia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
INTRODUCTION: Surgery is one of the treatments of choice for patients with a single metastasis from melanoma but is rarely curative. Such patients could potentially benefit from consolidation immunotherapy. Vaccination with dendritic cells (DCs) loaded with tumour antigens elicits a tumour-specific immune response. In our experience, patients who developed delayed type hypersensitivity (DTH) after DC vaccination showed a median overall survival (OS) of 22.9 monthsvs4.8 months for DTH-negative cases. A phase II randomised trial showed an advantage OS of a DC vaccine over a tumour cell-based vaccine (2-year OS 72% vs31%, respectively). Given that there is no standard therapy after surgical resection of single metastases, we planned a study to compare vaccination with DCs pulsed with autologous tumour lysate versus follow-up. METHODS AND ANALYSIS: This is a randomised phase II trial in patients with resected stage III/IV melanoma. Assuming a median relapse-free survival (RFS) of 7.0 months for the standard group and 11.7 months for the experimental arm (HR 0.60), with a two-sided tailed alpha of 0.10, 60 patients per arm must be recruited. An interim futility analysis will be performed at 18 months. The DC vaccine, produced in accordance with Good Manufacturing Practice guidelines, consists of autologous DCs loaded with autologous tumour lysate and injected intradermally near lymph nodes. Vaccine doses will be administered every 4 weeks for six vaccinations and will be followed by 3 million unit /day of interleukin-2 for 5 days. Tumour restaging, blood sampling for immunological biomarkers and DTH testing will be performed every 12 weeks. ETHICS AND DISSEMINATION: The protocol, informed consent and accompanying material given to patients were submitted by the investigator to the Ethics Committee for review. The local Ethics Committee and the Italian Medicines Agency approved the protocol (EudraCT code no.2014-005123-27). Results will be published in a peer-reviewed international scientific journal. TRIAL REGISTRATION NUMBER: 2014-005123-27.
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Vacunas contra el Cáncer , Extractos Celulares/uso terapéutico , Células Dendríticas/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Antineoplásicos/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Células Dendríticas/trasplante , Humanos , Interleucina-2/uso terapéutico , Melanoma/patología , Melanoma/terapia , Ensayos Clínicos Controlados Aleatorios como Asunto , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Trasplante AutólogoRESUMEN
Lenalidomide is a therapeutically effective drug in chronic lymphocytic leukemia (CLL). Twenty-seven CLL patients were treated with lenalidomide in a phase II clinical trial. Ten patients were grouped as responders (R) and 6 as nonresponders (NR). We evaluated T lymphocytes, NK, monocytes and dendritic cells at baseline and after treatment. A gene expression analysis was performed on 16 CLL samples collected before treatment. The levels of immune cells or immune-related cytokines are not different between R and NR patients. However, CLL patients sensitive to lenalidomide clearly show a peculiar gene expression profile in leukemic cells. The most up-regulated gene (fold change = +23 in R vs. NR) is Wnt inhibitor SHISA homolog 3 (SHISA3). SHISA3highCLL are characterized by a restrained activation of Wnt signaling and sensibility to lenalidomide-induced apoptosis. In conclusion, SHISA3 is a candidate gene for the identification of CLL patients who will benefit of lenalidomide treatment as single agent.
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Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Talidomida/análogos & derivados , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores de Tumor/metabolismo , Citocinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunomodulación/efectos de los fármacos , Lenalidomida , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Talidomida/farmacología , Talidomida/uso terapéuticoRESUMEN
Although immunomodulating antibodies are highly effective in metastatic melanoma, their toxicity, related to the activation of T lymphocytes, can be severe. Anticancer vaccines promote a fairly specific response and are very well tolerated, but their effectiveness has yet to be demonstrated. We have been treating patients with advanced melanoma with an autologous dendritic cell vaccine since 2001; to better characterize the safety and efficacy of our product, we designed a retrospective study on all of our patients treated with the vaccine to date. We retrospectively reviewed both case report forms of patients included in clinical trials and medical records of those treated within a compassionate use program. Response was assessed according to the Response Evaluation Criteria In Solid Tumors criteria and toxicity has been graded according to CTCAE 4.0. Although the response rate has been rather low, the median overall survival of 11.4 months and the 1-year survival rate of 46.9% are encouraging, especially considering the fact that data were obtained in a heavily pretreated population and only about one quarter of the patients had received ipilimumab and/or BRAF inhibitors. Multivariate analysis confirmed that the development of an immune response was significantly correlated with a better prognosis (hazard ratio 0.54; P=0.019). The adverse events observed were generally mild and self-limiting. Our analysis confirms the excellent tolerability of our vaccine, making it a potential candidate for combination therapies. As efficacy seems largely restricted to immunoresponsive patients, future strategies should aim to increase the number of these patients.
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Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Neoplasias Cutáneas/patología , Factores de Tiempo , Resultado del TratamientoRESUMEN
Lenalidomide is an IMID immunomodulatory agent clinically active in patients with chronic lymphocytic leukemia (CLL). We evaluated the activity of lenalidomide inside an in vitro coculture system of endothelial and CLL cells. Lenalidomide was able to inhibit CLL survival advantage mediated by endothelial contact. Moreover, the marked increase of in vitro angiogenesis determined by CLL-derived conditioned media was reduced by lenalidomide. We also analyzed peripheral blood collected from 27 patients with relapsed or refractory CLL being treated with lenalidomide within a phase II trial. Plasma levels of VEGF and THBS-1 decreased, whereas Ang2 and Ang increased during treatment. Patients who respond to lenalidomide showed a more pronounced decrease of VEGF and bFGF than did patients with stable or progressive disease (p = 0.007 and p = 0.005). Furthermore, lenalidomide reduced circulating endothelial cells and endothelial progenitors by increasing the percentage of apoptotic cells. Conversely, for six matched bone marrow biopsies available before and after treatment, we did not detect any modification in vessel density, suggesting a possible mechanism of vessel normalization rather than regression. In conclusion, our study provides further evidence that the anti-CLL effect of lenalidomide is mediated through the alteration of microenvironmental elements, implying the modulation of several angiogenesis-related factors and disruption of CLL crosstalk with endothelial cells.
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Inductores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Supervivencia Celular , Células Endoteliales/patología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Talidomida/análogos & derivados , Femenino , Humanos , Lenalidomida , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Talidomida/uso terapéuticoRESUMEN
The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ET(A)R and ETBR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ET(A)R axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ETA receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ET(A)R triggering. Moreover, the blockade of ET(A)R by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ET(A)R via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ET(A)R blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ET(A)R signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ET(A)R inhibition.
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Resistencia a Antineoplásicos , Endotelina-1/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Receptor de Endotelina A/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Antagonistas de los Receptores de la Endotelina A/farmacología , Endotelina-1/sangre , Endotelina-1/genética , Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Pronóstico , Receptor de Endotelina A/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Resultado del TratamientoRESUMEN
Increasing evidence suggests a key role for angiopoietin-2 (ANGPT2) in influencing the aggressiveness of chronic lymphocytic leukemia (CLL). In the presence of vascular endothelial growth factor (VEGF), ANGPT2 causes vessel destabilization leading to neoangiogenesis. Accordingly, high expression levels of ANGPT2 and high degree of angiogenesis have consistently been associated with poor prognosis in CLL; however, the molecular mechanisms behind the variability in ANGPT2 expression are still to be discovered. Here, for the first time, we investigated the DNA methylation status of the ANGPT2 promoter in a large CLL cohort (n = 88) using pyrosequencing and correlated methylation data with ANGPT2 expression levels, prognostic factors and outcome. Importantly, methylation levels of the ANGPT2 gene correlated inversely with its mRNA expression levels (p<0.001). Moreover, low ANGPT2 methylation status was highly associated with adverse prognostic markers, shorter time to first treatment and overall survival. Finally, treatment with methyl inhibitors induced re-expression of ANGPT2 in two B-cell lymphoma cell lines, underscoring the importance of DNA methylation in regulating transcriptional silencing of this gene. In conclusion, we believe that the known variability in ANGPT2 expression among CLL patients could be explained by differential promoter DNA methylation and that low methylation levels of the ANGPT2 promoter have an adverse prognostic impact in CLL.