Lisa Villabona

HLA-dependent tumour development: a role for tumour associate macrophages?

HLA abnormalities on tumour cells for immune escape have been widely described. In addition, cellular components of the tumour microenvironment, in particular myeloid derived suppressor cells (MDSC) and alternatively activated M2 tumour-associated macrophages (TAMs), are involved in tumour promotion, progression, angiogenesis and suppression of anti-tumour immunity. However, the role of HLA in these activities is poorly understood. This review details MHC class I characteristics and describes MHC class I receptors functions. This analysis established the basis for a reflection about the crosstalk among the tumour cells, the TAMs and the cells mediating an immune response.The tumour cells and TAMs exploit MHC class I molecules to modulate the surrounding immune cells. HLA A, B, C and G molecules down-regulate the macrophage myeloid activation through the interaction with the inhibitory LILRB receptors. HLA A, B, C are able to engage inhibitory KIR receptors negatively regulating the Natural Killer and cytotoxic T lymphocytes function while HLA-G induces the secretion of pro-angiogenic cytokines and chemokine thanks to an activator KIR receptor expressed by a minority of peripheral NK cells. The open conformer of classical MHC-I is able to interact with LILRA receptors described as being associated to the Th2-type cytokine response, triggering a condition for the M2 like TAM polarization. In addition, HLA-E antigens on the surface of the TAMs bind the inhibitory receptor CD94/NKG2A expressed by a subset of NK cells and activated cytotoxic T lymphocytes protecting from the cytolysis.Furthermore MHC class II expression by antigen presenting cells is finely regulated by factors provided with immunological capacities. Tumour-associated macrophages show an epigenetically controlled down-regulation of the MHC class II expression induced by the decoy receptor DcR3, a member of the TNFR, which further enhances the M2-like polarization. BAT3, a positive regulator of MHC class II expression in normal macrophages, seems to be secreted by TAMs, consequently lacking its intracellular function, it looks like acting as an immunosuppressive factor.In conclusion HLA could cover a considerable role in tumour-development orchestrated by tumour-associated macrophages.

Correlation of HLA-A02* genotype and HLA class I antigen down-regulation with the prognosis of epithelial ovarian cancer

BackgroundIn recent years, evidence is accumulating that cancer cells develop strategies to escape immune recognition. HLA class I HC down-regulation is one of the most investigated. In addition, different HLA haplotypes are known to correlate to both risk of acquiring diseases and also prognosis in survival of disease or cancer. We have previously shown that patients with serous adenocarcinoma of the ovary in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A02* genotype. We aimed to study the relationship between HLA-A02* genotype in these patients and the subsequent HLA class I HC protein product defects in the tumour tissue.Materials and methodsOne hundred and sixty-two paraffin-embedded tumour lesions obtained from Swedish women with epithelial ovarian cancer were stained with HLA class I heavy chain (HC) and β2-microglobulin (β2-m)-specific monoclonal antibodies (mAb). Healthy ovary and tonsil tissue served as a control. The HLA genotype of these patients was determined by PCR/sequence-specific primer method. The probability of survival was calculated using the Kaplan–Meier method, and the hazard ratio (HR) was estimated using proportional hazard regression.ResultsImmunohistochemical staining of ovarian cancer lesions with mAb showed a significantly higher frequency of HLA class I HC and β2-m down-regulation in patients with worse prognosis (WP) than in those with better prognosis. In univariate analysis, both HLA class I HC down-regulation in ovarian cancer lesions and WP were associated with poor survival. In multivariate Cox-analysis, the WP group (all with an HLA-A02* genotype) had a significant higher HR to HLA class I HC down-regulation.ConclusionsHLA-A02* is a valuable prognostic biomarker in epithelial ovarian cancer. HLA class I HC loss and/or down-regulation was significantly more frequent in tumour tissues from HLA-A02* positive patients with serous adenocarcinoma surgical stage III–IV. In multivariate analysis, we show that the prognostic impact is reasonably correlated to the HLA genetic rather than to the expression of its protein products.

Non-classical HLA-class I expression in serous ovarian carcinoma: Correlation with the HLA-genotype, tumor infiltrating immune cells and prognosis.

In our previous studies, we have shown that patients with serous ovarian carcinoma in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A*02 genotype. This represent a stronger prognostic factor than loss or downregulation of the MHC class I heavy chain (HC) on tumor cells. In this study, we investigated the expression of the non-classical, immune tolerogenic HLA -G and -E on the tumor cells along with the infiltration of immune cells in the tumor microenvironment. FFPE primary tumors from 72 patients with advanced stages of serous adenocarcinoma and metastatic cells present in ascites fluid from 8 additional patients were included in this study. Both expression of HLA-G and aberrant expression of HLA-E were correlated to a significant worse prognosis in patients with HLA-A*02, but not with different HLA genotypes. Focal cell expression of HLA-G correlated to a site-specific downregulation of classical MHC class I HC products and aberrant HLA-E expression, showing a poor survival. HLA-G was more frequently expressed in metastatic cells than in primary tumor lesions and the expression of HLA-G inversely correlated with the frequency of tumor infiltrating immune cells. All these parameters can contribute together to identify and discriminate subpopulations of patients with extremely poor prognosis and can give them the opportunity to receive, and benefit of individually tailored treatments.

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

The aim of this study was to establish a novel approach for human leukocyte antigen (HLA)-typing from formalin-fixed paraffin-embedded-derived DNA. HLAs can be a prognostic factor in cancer and have an extensive polymorphism. This polymorphism is predominantly restricted to exons, which encode the peptide-binding domain of the protein. Formalin-fixed paraffin-embedded material is routinely collected in the clinic and therefore a great source of DNA for genetic analyses. However, its low quality due to fragmentation and nucleotide changes has often created obstacles in designing genetic assays. In this study, we amplified the most polymorphic exons of the HLA-A gene, exons 2, 3, and 4, in 16 formalin-fixed paraffin-embedded samples >10 years old. These tissue samples belonged to patients already HLA-typed by peripheral blood samples at the routine laboratory. Acquired amplification products were used for sequencing, which provided enough information to establish an HLA allele. The same method was applied to DNA extracted from peripheral blood from a healthy volunteer with known HLA type. Of the samples, 14/16 (88%) were successfully typed, in one sample only one of the alleles could be determined, and in one sample no allele could be determined. The amplification of the most polymorphic exons of HLA-A was a successful alternative when DNA quality prevented positive results with previously described methods. The method is usable when an HLA type is needed but the patients are deceased and/or no whole blood samples can be collected. It has thus potential to be used in several fields such as the clinic, research, and forensic science.

Genotyping of Human Leukocyte Antigen (HLA) Ancestral Haplotypes as Prognostic Marker in Cancer Using PCR Analysis

The major histocompatibility complex (MHC) comprises a set of genes that are essential to immunity and surveillance against neoplastic transformation. MHC antigens not only regulate antitumor immune responses in experimental animal models but also directly correlate with survival and prognosis of patients with various types of cancers. Effective recognition of tumor cells by effector T cells may be affected by the genotype and the extent of expression of human leukocyte antigen (HLA)-peptide complexes. Therefore, MHC antigens may serve as potential biomarkers for prognosis and allow selection of cancer patients for specific therapy. We describe PCR-based method to determine the HLA genotype in healthy individuals and patients using blood and tumor tissue as DNA source.

Survival rates in HLA-A2 positive patients, with stage III-IV serous adenocarcinomas of the ovary, correlates with increased expression of COX-2 and iNOS, loss of MHC Class I and II and defects of the antigen processing mechanisms at the tumour level

16544 Background: We have shown that the high prevalence of HLA-A2 in Scandinavia is associated to poor prognosis in advanced (stage III-IV) serous adenocarcinoma of the ovary. Our objective was to analyse whether there exists a relationship between the expression of HLA molecules and components of the antigen processing pathway as well as the presence of high COX-2 activity and the patients´ survival. Methods: To test our hypothesis, 40 Swedish women with epithelial ovarian cancer were selected from an unbiased database of ovarian cancer patients at our department. Their HLA haplotypes were defined by the PCR/sequence-specific oligonucleotide hybridization method (PCR/SSOP) Tissue slides from paraffin embedded tumour material were stained with antibodies recognising COX-2, iNOS, MHC-class I/II heavy chain (HC) and molecules of the antigen processing machinery (APM). The probability of survival was defined by Kaplan-Meier analysis. Results: HLA-A2 positive patients, in stage III-IV were distinguished with...