Rafael Carretero

Analysis of HLA class I expression in progressing and regressing metastatic melanoma lesions after immunotherapy

Despite the potential efficacy of cancer immunotherapy in preclinical studies, it did not show yet significant positive clinical results in humans with only a small number of cancer patients demonstrating objective tumor regression. This poor clinical outcome can be explained by the generation of sophisticated tumor immune escape mechanism, in particular, abnormalities in the expression of HLA class I antigens. We have studied the expression of HLA class I antigens in ten metastatic lesions obtained from a melanoma patient undergoing immunotherapy. Five lesions were obtained after Interferon-alpha-2b treatment and five after autologous vaccination plus BCG (M-VAX). Eight metastases were regressing after immunotherapy while two were progressing. The eight regressing metastases showed high level of HLA class I expression, whereas the two progressing lesions had low levels as measured by real time PCR and immunohistological techniques. These results indicate a strong association between HLA class I expression and progression or regression of the metastatic lesions. Our data support the hypothesis that the level of HLA class I expression is an important parameter of tumor immune escape that needs to be monitored.

Regression of melanoma metastases after immunotherapy is associated with activation of antigen presentation and interferon-mediated rejection genes

We present the results of a comparative gene expression analysis of 15 metastases (10 regressing and 5 progressing) obtained from 2 melanoma patients with mixed response following different forms of immunotherapy. Whole genome transcriptional analysis clearly indicate that regression of melanoma metastases is due to an acute immune rejection mediated by the upregulation of genes involved in antigen presentation and interferon mediated response (STAT‐1/IRF‐1) in all the regressing metastases from both patients. In contrast, progressing metastases showed low transcription levels of genes involved in these pathways. Histological analysis showed T cells and HLA‐DR positive infiltrating cells in the regressing but not in the progressing metastases. Quantitative expression analysis of HLA‐A,B and C genes on microdisected tumoral regions indicate higher HLA expression in regressing than in progressing metastases. The molecular signature obtained in melanoma rejection appeared to be similar to that observed in other forms of immune‐mediated tissue‐specific rejection such as allograft, pathogen clearance, graft versus host or autoimmune disease, supporting the immunological constant of rejection. We favor the idea that the major factor determining the success or failure of immunotherapy is the nature of HLA Class I alterations in tumor cells and not the type of immunotherapy used. If the molecular alteration is reversible by the immunotherapy, the HLA expression will be upregulated and the lesion will be recognized and rejected. In contrast, if the defect is structural the MHC Class I expression will remain unchanged and the lesion will progress.

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer

BackgroundInflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk.MethodsA case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area.ResultsDiagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09–2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09–2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms.ConclusionOur results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.

Regressing and progressing metastatic lesions: resistance to immunotherapy is predetermined by irreversible HLA class I antigen alterations

Despite the significant efforts to enhance immune reactivity against malignancies the clinical effect of anti-tumor vaccines and cancer immunotherapy is still below expectations. Understanding of the possible causes of such poor clinical outcome has become very important for improvement of the existing cancer treatment modalities. In particular, the critical role of HLA class I antigens in the success of T cell based immunotherapy has led to a growing interest in investigating the expression and function of these molecules in metastatic cancer progression and, especially in response to immunotherapy. In this report, we illustrate that two types of metastatic lesions are commonly generated in response to immunotherapy according to the pattern of HLA class I expression. We found that metastatic lesions, that progress after immunotherapy have low level of HLA class I antigens, while the regressing lesions demonstrate significant upregulation of these molecules. Presumably, immunotherapy changes tumor microenvironment and creates an additional immune selection pressure on tumor cells. As a result, two subtypes of metastatic lesions arise from pre-existing malignant cells: (a) regressors, with upregulated HLA class I expression after therapy, and (b) progressors with resistance to immunotherapy and with low level of HLA class I. Tumor cells with reversible defects (soft lesions) respond to therapy by upregulation of HLA class I expression and regress, while tumor cells with structural irreversible defects (hard lesions) demonstrate resistance to immunostimulation, fail to upregulate HLA class I antigens and eventually progress. These two types of metastases appear independently of type of the immunotherapy used, either non-specific immunomodulators (cytokines or BCG) or autologous tumor vaccination. Similarly, we also detected two types of metastatic colonies in a mouse fibrosarcoma model after in vitro treatment with IFN-γ. One type of metastases characterized by upregulation of all MHC class I antigens and another type with partial IFN-γ resistance, namely with lack of expression of Ld-MHC class I molecule. Our observations may shed new light on the understanding of the mechanisms of tumor escape and might have implications for improvement of the efficacy of cancer immunotherapy.

Impact of interleukin-18 polymorphisms-607 and -137 on clinical characteristics of renal cell carcinoma patients

Current evidence suggests that chronic inflammation is associated with tumor development and progression. Interleukin-18 (IL-18) plays a central role in inflammation and the immune response, contributing to the pathogenesis and pathophysiology of infectious and inflammatory diseases. The objective of this study was to determine whether the presence of IL-18 polymorphisms -137 G/C (rs187238) and -607 A/C (rs1946518) was associated with size, grade, TNM stage, and survival in patients with renal cell carcinoma (RCC). The study cohort included 158 patients with RCC. Control group consisted of 506 samples from Spanish population. The studied IL-18 gene polymorphisms did not influence susceptibility to RCC in the analyzed group of patients (IL-18-607, p = 0.318; IL-18-137 p = 0.740) but may contribute to disease onset and aggressiveness. IL-18-607 CC genotype was significantly associated with higher tumor size (p = 0.001), grade (p = 0.030), T (p = 0.001), M (p = 0.012), and stage (p = 0.002). IL-18-103 GG genotype was correlated with higher tumor size (p = 0.036), grade (p = 0.017), T (p = 0.026), and stage (p = 0.011). The Cox proportional hazard model showed that nuclear grade and stage grouping were independent prognostic factors but IL-18 polymorphism was not. Polymorphism variants in the IL-18 gene (IL-18-607 and IL-18-137) may be associated with a worse prognosis for RCC. High levels of IL-18 production may play a major role in the growth, invasion and metastasis of renal cancer.

Bacillus Calmette‐Guerin immunotherapy of bladder cancer induces selection of human leukocyte antigen class I‐deficient tumor cells

Bacillus Calmette‐Guerin (BCG) immunotherapy is a standard treatment for high‐risk non‐muscle‐infiltrating bladder cancer patients. Although the outcomes are good, cancer relapse is observed in around 40% of patients. We present the comparative analysis of human leukocyte antigen (HLA) class I expression in recurrent bladder tumors in patients treated with mitomycin or BCG. HLA class I expression was analyzed by RT‐Q‐PCR and immunohistochemical techniques. Loss of heterozygosity (LOH) was determined by microsatellite amplification of markers in chromosome 6 and 15. More profound alterations in HLA class I expression were found in post‐BCG recurrent tumors than in pre‐BCG lesions, whereas mitomycin treatment did not change the HLA class I expression pattern. Post‐BCG recurrent tumors also showed a higher incidence of structural defects underlying altered HLA class I expression. We hypothesize that the immunotherapy‐activated immune system recognizes and eliminates tumor cells with reversible (“soft”) HLA class I changes but not transformed cells with additional, irreversible (“hard”) alterations. To our knowledge, this is the first clinical evidence of immunotherapy‐induced immunoselection of HLA class I loss tumor variants in bladder cancer, although the study involved a small number of patients.

Association between C13ORF31, NOD2, RIPK2 and TLR10 polymorphisms and urothelial bladder cancer

Several evidences have been published linking polymorphism in genes involved in chronic or recurrent inflammation with increased tumor risk and progression. Nevertheless the influence of innate immune receptors in urothelial cancer risk and characteristics has not been sufficient explored. We studied the possible association of polymorphisms in genes encoding NOD2, RIPK2, TLR10 and C13ORF31 with the risk, clinical/pathological characteristics and outcomes of urothelial cancer. We have found association between RIPK2 (rs42490) and cancer risk (AA vs AT&TT, p=0042). In addition, we found statistical differences in TLR10 (rs4129009) gen between low and high tumor infiltration stage (p=0.033). NOD2 (rs9302752) and RIPK2 (rs42490) were found to be associated with development of lymph node metastasis (p=0.011 and p=0.015). Importantly we detect association of TLR10 (Log Rank=0.035) and RIPK2 (Log Rank=0040) with overall survival. Multivariate Cox analysis revealed that both SNPs were survival prognosis factor independent of tumor stage and grade. Our results indicate that innate immunity receptors play a role in modulating urothelial cancer risk and progression.

Changes in activatory and inhibitory natural killer (NK) receptors may induce progression to multiple myeloma: implications for tumor evasion of T and NK cells.

The molecular basis of monoclonal gammopathy of undetermined significance (MGUS) progression to a malignant monoclonal gammopathy remains poorly understood. It was recently suggested that this process involves the suppression of innate and adaptive immunity. In this study, we examined immunogenic differences in bone marrow plasma cells among individuals without gammopathy (controls) and patients with MGUS, multiple myeloma (MM), and plasma cell leukemia. We detected differences in major histocompatibility complex (MHC) class I expression, MHC class I chain-related molecule A, and CD95 that were more evident between MGUS and MM samples; there appeared to be a critical imbalance between natural killer (NK)-cell activating and inhibitory signals during the transition from MGUS to MM. Our results indicate that the human leukocyte antigen (HLA) class I(bright), MICA(dim/-), and CD95(dim/-) immunophenotype reported in myeloma cells may result from an extensive interaction of malignant cells with cytotoxic T and NK cells and appears to be immunoedited for the evasion of immunosurveillance.

A Transcriptome-proteome Integrated Network Identifies Endoplasmic Reticulum thiol oxidoreductase (ERp57) as a Hub that Mediates Bone Metastasis

Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared with parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein-protein interaction network and differential expression. The protein-protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub that retained four down-regulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E, and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.

VEGF polymorphisms are not associated with an increased risk of developing renal cell carcinoma in Spanish population

PURPOSE Vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis and is over-expressed in renal cell cancer (RCC). Published data on the association between polymorphisms of vascular endothelial growth factor (e.g., -2578C/A [rs699947], -460T/C [rs833061], +405C/G [rs2010963], and +936C/T [rs3025039]) and the risk of renal cell carcinoma are ambiguous and controversial. The aim of this investigation was to investigate this relationship in a series of Caucasian Spanish patients. MATERIALS AND METHODS A case-control study was performed with 216 cases and 280 controls, genotyping subjects for VEGF polymorphisms using the predesigned TaqMan single nucleotide polymorphism (SNP) genotyping assay (Applied Biosystems, Foster City, CA, USA). The combined effect of the four gene polymorphisms on overall survival was studied by haplotype analysis. RESULTS The overall results suggest that polymorphisms or haplotypes in the VEGF gene do not modify the risk of RCC. We were unable to replicate the association of the -460T/C (rs833061) polymorphism with renal cancer risk. Data were also gathered on clinical-pathological results, tumor size, clinical stage, histological grade, and survival. CONCLUSIONS According to our analysis of their contribution to prognostic factors, VEGF polymorphisms do not appear to exert a significant influence on RCC progression or prognosis. This finding might be explained by the tumor biology and pathogenesis of clear cell RCC. Additional studies with larger sample sizes are needed in different ethnic groups to further assess this association.