Isabel Maleno

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.

Multiple mechanisms generate HLA class I altered phenotypes in laryngeal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

Abstract.Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.

Distribution of HLA class I altered phenotypes in colorectal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

HLA class I loss or down-regulation is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes, and thus favor tumor immune escape. Multiple mechanisms are responsible for these HLA class I alterations. In different epithelial tumors, loss of heterozygosity (LOH) at chromosome region 6p21.3, leading to HLA haplotype loss, occurs in 6–50% of all cases depending on the tumor entity. In this paper we report the frequency of LOH at 6p21 in 95 colorectal carcinomas (CRC) previously analyzed for altered HLA class I expression with immunohistological techniques. We used PCR microsatellite amplification of selected STR markers located on Chromosome 6 to identify LOH with DNA from microdissected tumor tissues and the surrounding stroma. Sequence-specific oligonucleotide analysis was performed in microdissected stroma and tumor cells for HLA typing, and to detect HLA haplotype loss. A high frequency (40%) of HLA haplotype loss was found in CRC. Eight tumors showed microsatellite instability. We sometimes observed two or more mechanisms responsible for HLA alteration within the same HLA-altered phenotype, such as LOH and HLA class I total loss. In 25 tumors (26%) no HLA class I alteration could be identified. These data are potentially relevant for CRC patients undergoing T-cell-based immunotherapy.

HLA class I expression in metastatic melanoma correlates with tumor development during autologous vaccination

Our knowledge of the mechanisms underlying tumor-specific immune response and tumor escape has considerably increased. HLA class I antigen defects remain an important tumor escape mechanism since they influence the interactions between tumor cells and specific T and NK cells in the course of malignant disease. We have studied here HLA class I expression in six subcutaneous metastases obtained from a melanoma patient immunized with an autologous melanoma cell vaccine (M-VAX). We report in this paper that HLA class I antigen expression on these metastatic lesions strongly correlated with the course of the disease. The three metastases that were partially regressing at the time of their excision showed a strong HLA class I expression, whereas the progressing ones showed a very weak or negative staining with most of the anti-HLA class I mAbs used. Real-time quantitative PCR of the samples obtained from microdissected tumor tissue revealed a significant difference in the mRNA levels of HLA-ABC heavy chain and β2m between the two types of metastases, i.e., lower levels in progressing metastases and high levels in regressing ones, confirming the immunohistological findings. This is, to our knowledge, the first report where the clinical outcome of different HLA class I positive and negative melanoma metastases can be clearly correlated with the regression and progression of the disease, respectively.

Molecular strategies to define HLA haplotype loss in microdissected tumor cells

Loss of heterozygosity (LOH) of chromosome 6p21 is an important mechanism that generates HLA haplotype loss in various human tumors. This mechanism produces non-reversible HLA-deficient tumor cells that can escape T cell immune responses in peptide-vaccinated cancer patients. However, the exact frequency of this mechanism is still unknown, because contaminating stroma in solid tumor tissues masks the tumor DNA obtained from solid samples. A microdissection technique was applied to 4-8 microm sections of cryopreserved tumor tissues from a group of colorectal and laryngeal carcinomas. Fifteen patients were analyzed for the presence of LOH associated with the beta(2)-microglobulin gene in chromosome 15, and five patients for LOH associated with HLA genes in chromosome 6. In two cases, autologous metastasis tissue samples were also available. The patients were selected for showing an altered HLA class I tumor phenotype as determined by immunohistological techniques. DNA was obtained from this microdissected material and amplified in order to detect the presence or absence of nine previously selected microsatellite markers. HLA sequence based typing (SBT) was also applied to these microdissected DNA samples to define the HLA genotype. Microdissection greatly improved the definition of LOH, with nearly 100% signal reduction in one of the alleles. In addition, this procedure allowed us to detect beta(2)-microglobulin LOH in tumors that expressed some HLA molecules. Our data indicate that this procedure can be successfully applied to microdissected samples from solid tumors, thus enhancing the power and sensitivity of LOH detection.

HLA and melanoma: multiple alterations in HLA class I and II expression in human melanoma cell lines from ESTDAB cell bank.

Altered HLA class I and class II cell surface expression has been reported in many types of malignancy and represents one of the major mechanism by which tumour cells escape from T lymphocytes. In this report, we review the results obtained from the study of constitutive and IFN-gamma-induced expression of HLA class I and II molecules in 91 human melanoma cell lines from the European Searchable Tumour Cell Line Database, and compare them with published data on HLA expression in other types of cancer. Various types of alterations in HLA class I cell surface expression were found in a high percentage (67%) of the studied cell lines. These alterations range from total to selective HLA class I loss and are associated with β2-microglobulin gene mutations, transcriptional downregulation of HLA class I genes and antigen processing machinery components, or with the loss of heterozygosity in chromosome 6. The most frequently observed phenotype is selective downregulation of HLA-B locus, reversible after treatment with IFN-gamma. The expression of constitutive- or IFN-gamma induced-surface expression of at least one HLA class II locus is positive in 71.5% of the analysed cell lines. Four different HLA class II expression phenotypes were defined, and a positive correlation between the expression of class I and II molecules is discussed. More detailed information on the HLA expression patterns and others immunological characteristics of these melanoma cell lines can be found on the following website http://www.ebi.ac.uk/ipd/estdab.

Characterization of HLA class I altered phenotypes in a panel of human melanoma cell lines

BackgroundAltered HLA class I cell surface expression is one of the major mechanisms by which tumor cells escape from T lymphocytes. Immunohistochemistry-defined phenotypes of lost HLA class I expression have been described in human solid tumors, nut less information is available on melanoma cell lines.ObjectivesTo describe the frequency and distribution of different types of HLA class I antigen alterations in 91 melanoma cell lines from the European Searchable Tumour Cell and Databank (ESTDAB).MethodsThe HLA class I expression was assessed by flow cytometry and HLA genotyping.ResultsWe found various types of HLA class I cell surface alterations in about 67% of the melanoma cell lines. These alterations range from total to selective HLA class I loss due to loss of heterozygosity (LOH), haplotype loss, β2-microglobulin gene mutation, and/or total or selective down-regulation of HLA class I molecules. The most frequently observed phenotype is down-regulation of HLA-B locus that was reversible after treatment with IFN -γ.ConclusionsIn general, HLA class I alterations in the majority of the cells analyzed were of regulatory nature and could be restored by IFN-γ. Analysis of the frequency of distinct HLA class I altered phenotypes in these melanoma cell lines revealed specific differences compared to other types of tumors.

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors.

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.

LOH at 6p21.3 region and HLA class I altered phenotypes in bladder carcinomas

Alterations in HLA class I antigen expression have been frequently described in different epithelial tumors and are thought to favor tumor immune escape from T lymphocyte recognition. Multiple molecular mechanisms are responsible for these altered HLA class I tumor phenotypes. Some are structural defects that produce unresponsiveness to treatment with interferons. Others include alterations in regulatory mechanisms that can be switched on by treatment of tumor cells with different cytokines. One important mechanism belonging to the first group is loss of heterozygosity (LOH) at chromosome region 6p21.3, which can lead to HLA haplotype loss. In this investigation, the frequency of LOH at 6p21 chromosome region was studied in 69 bladder carcinomas. Short tandem repeat analysis showed that 35% of cases had LOH in this chromosome region. By considering these results together with immunohistological findings previously published by our group, we identified a distribution pattern of HLA class I altered phenotypes in bladder cancer. The most frequently altered phenotype in bladder carcinomas was total loss of HLA class I expression (17 cases, 25%), followed by phenotype II associated with HLA haplotype loss (12 cases, 17.5%), and HLA allelic loss (ten cases, 14.5%). Nine cases (13%) were classified as having a compound phenotype, five cases (7%) as having HLA locus loss, and in 16 cases (23%) no alteration in HLA expression was detected. An important conclusion of this report is that a combination of different molecular and immunohistological techniques is required to precisely define which HLA alleles are lost during tumor progression and to characterize the underlying mechanisms of these losses. These studies should be performed when a cancer patient is to be included in an immunotherapy protocol that aims to stimulate different immune effector mechanisms.

High frequency of homozygosity of the HLA region in melanoma cell lines reveals a pattern compatible with extensive loss of heterozygosity

Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. MHC class I loss or down-regulation in cancer cells is a major immune escape route used by a large variety of human tumours to evade antitumour immune responses mediated by cytotoxic T lymphocytes. The goal of our study was to explore HLA genotyping and phenotyping in a variety of melanoma tumour cell lines. A total of 91 melanoma cell lines were characterised for HLA class I and II genotype. In addition, 61 out of the 91 cell lines were also analysed for HLA class I and II cell surface molecule expression by flow cytometry. Unexpectedly, we found that 19.7% of the melanoma cell lines were homozygous for HLA class I genotypes, sometimes associated with HLA class II homozygosity (8.79%) and sometimes not (10.98%). The frequency of homozygosity was significantly higher compared with the control groups (1.6%). To identify the reasons underlying the high frequency of HLA homozygosity we searched for genomic deletions using eight pairs of highly polymorphic microsatellite markers covering the entire extended HLA complex on the short arm of chromosome 6. Our results were compatible with hemizygous deletions and suggest that loss of heterozygosity on chromosome arm 6p is a common feature in melanoma cell lines. In fact, although autologous normal DNA from the patients was not available and could not be tested, the retention in some cases of heterozygosity for a number of microsatellite markers would indicate a hemizygous deletion. In the rest of the cases, markers at 6p and 6q showed a single allele pattern indicating the probable loss of part or the whole of chromosome 6. These results led us to conclude that loss of heterozygosity in chromosome 6 is nonrandom and is possibly an immunologically relevant event in human malignant melanoma. Other well-established altered HLA class I phenotypes were also detected by flow cytometry that correspond to HLA class I total loss and HLA-ABC and/or specific HLA-B locus down-regulation.