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

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.     

LOH at 6p21.3 region and HLA class 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.     

Expression of glucose transporter 1 is associated with loss of heterozygosity of chromosome 1p in oligodendroglial tumors WHO grade II

Objective Oligodendroglial tumors with a loss of heterozygosity of 1p (LOH1p) appear to have a better prognosis than oligodendrogliomas without LOH. Previously, we reported glucose uptake in low-grade oligodendroglial tumors to be related to LOH 1p status. Here, we performed an immunohistochemical study of the common glucose transporters (GLUT) in relation to LOH1p. Material and methods We examined 17 oligodendrogliomas (O II, 11 with LOH1p), 16 oligoastrocytomas (OA II, 5 with LOH1p) and 7 astrocytomas (A II, none with LOH1p). Confocal microscopy was performed for p53, GLUT-1, −3 and −12. Immunoreaction was rated semiquantitatively by percentage of positive cells and staining intensity on immunohistological stainings. Results Confocal microscopy depicted immunoreaction for GLUT-1, −3 and −12 in the cytoplasm of the tumor cells. Oligodendrogliomas revealed a lower immunoreactivity for GLUT-1 than oligoastrocytomas and astrocytomas (P = 0.0263). No differences in immunoreactivity were found for GLUT-3 and GLUT-12. GLUT-1 expression in tumors with LOH 1p was significantly lower than in tumors with wild type 1p status (P = 0.0017). GLUT-3 and GLUT-12 immunoreactivity was not correlated with LOH 1p. Conclusion Expression of GLUT-1 is significantly reduced in low-grade oligodendroglial tumors harboring LOH 1p. Further studies should address the functional role of GLUT-1 in regard to chemosensitivity of oligodendrogliomas.     

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

Background Altered 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. Objectives To 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). Methods The HLA class I expression was assessed by flow cytometry and HLA genotyping. Results We 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. Conclusions In 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. Rosa Méndez and Teresa Rodríguez have equally contributed to this work and both should be considered as first authors.     

贲门癌中染色体8p21-p23杂合性丢失的研究

目的 研究贲门癌中染色体8p21-p23杂合性丢失的情况。方法 采用激光捕获显微切割技术获得均质的肿瘤细胞及正常的胃粘膜细胞,多重置换扩增技术扩增捕获细胞的基因组DNA。PCR结合硝酸银染色方法分析19例贲门癌染色体8p21-p23的杂合性丢失。结果 在贲门癌中染色体8p21-p23的缺失频率非常高（63.2%）,我们确定了一个最小丢失区域. 结论 进一步明确此最小丢失区域内的抑癌基因将有助于贲门癌发生机制的阐明。     

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.     

人类恶性肿瘤中染色体17p13.3区带的杂合性丢失

人类恶性肿瘤中经常发生染色体染合性丢失,从而丢失抑癌基因的某一个等基因。人类１７号染色本特别是该色体的１７ｐ１３．３区带,在多种肿瘤中都存在着染色的体杂支失。     

Loss of heterozygosity at chromosome 6 as a marker of early genetic alterations in cervical intraepithelial neoplasias and microinvasive carcinomas

Oncogenic human papillomaviruses (mostly HPV types 16 and 18) are the major cause of cervical intraepithelial neoplasia (CIN), which progresses into cervical cancer (CC). To reveal early genetic alterations of chromosome 6 that are important for CC progression, we analyzed the loss of heterozygosity (LOH) in DNAs from 45 CIN cases, 47 microcarcinomas, and 19 invasive squamous cell carcinomas stage IB. LOH analysis of DNA samples prepared with microdissection from all CIN foci, as well as from CC lesions and synchronous CIN, permitted investigation of CIN and CC heterogeneity. Out of all CC stage I cases, 79% showed LOH with six microsatellite markers at chromosome 6. LOH with the microsatellite markers D6S276 (6p22) and TNFa (6p21.3) was found in 50% of the CC cases. LOH frequency in CIN lesions synchronous with CC was higher then in CIN cases without cancer; the statistical significance (P = 0.004) was shown for D6S291 (6p21.2). The finding suggests that the high frequency of LOH in CIN lesions is a marker of unfavorable prognosis for CIN. Progression from microcarcinoma to invasive CC of stage IB was associated with a higher LOH frequency at D6S344 (6p25) and TNFa (6p21.3). Early genetic alterations were found in CIN with microsatellites D6S273 and TNFa located at 6p21.3. Moreover, LOH frequency at D6S273 remained the same in both CIN and CC cases. Based on HPV typing, LOH analysis, and X-chromosome inactivation, the polyclonality of CC lesions, as well as CIN, was observed in a few patients.     

Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots

Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK⁻ mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation. We then made use of an additional 17 microsatellite markers which provided an average map-interval of 1.6 Mb to map various LOH endpoints on the 45 Mb portion of chromosome 17q corresponding to the maximum length of LOH tracts (i.e. from the distal marker D17S932 to the terminal end). There appeared to be four prominent peaks (I–IV) in the distribution of LOH endpoints/Mb of Tk6-20C cells that were not evident in p53-abrogated cells TK6-E6, where they appeared to be rather broadly distributed along the 15–20 Mb length (D17S1807 to D17S1607) surrounding two of the peaks that we detected in TK6-20C cells (peaks II and III). We suggest that the chromosomal instability that is so evident in TK6-E6 cells may be due to DNA double-strand break repair occurring through non homologous end-joining rather than allelic recombination.     

Microsatellite instability analysis in tumors with different mechanisms for total loss of HLA expression

Down-regulation of the expression of major histocompatibility complex molecules is a frequent event that is associated with the poor immunogenicity of tumor cells. Acquired resistence to T-cell-based immunotherapy has been associated with loss of functional β2-microglobulin expression. This anomaly appears to be particularly relevant in tumors exhibiting a defect in DNA-mismatch repair, and induces structural abnormalities in HLA cell-surface expression that are not reversible by cytokine treatment. We examined HLA expression in 118 melanoma, colon or larynx tumors to identify total loss of HLA class I expression with or without somatic β2-microglobulin gene mutation. Microsatellite instability was investigated in these tumors to determine whether a replication error phenotype (RER⁺) implied a particular alteration in HLA phenotype. A total of 7.6% of the tumors showed the RER⁺ phenotype, and 12.7% were HLA-ABC-negative. In the RER⁺ group, only one tumor was HLA-ABC-negative and no β2-microglobulin mutation was identified. In contrast, in the HLA-ABC-negative group, only one tumor showed microsatellite instability. None of the three melanomas that contained β2-microglobulin mutation exhibited the mutator phenotype. These findings suggest that β2-microglobulin mutation in human melanoma tumors may arise through a mechanism that does not necessarily involve microsatellite instability. Our results also indicate that somatic mutations of the β2-microglobulin gene are not the main mechanism of total loss of HLA expression. Received: 14 June 1999 / Accepted: 16 September 1999     

Cytotoxic susceptibility and defective MHC class I expression do not correlate with mutation of p53 in human carcinomas

Twenty-nine samples of ex vivo ovarian and lung carcinomas were investigated for the relationship between the presence of mutated protein 53 (mp53) and cytotoxic susceptibility. Unaltered expression of MHC class I alleles was required for the cytotoxic susceptibility of tumour cells to the autologous ex vivo blood lymphocytes, i.e. all 4 sensitive tumours belonged to the group of 11 tumours without defect in MHC class I expression. In contrast, the susceptibility did not correlate with the presence of mp53, i.e. cases with mp53 were randomly distributed between the sensitive and resistant tumours (2/4 and 10/17 respectively). There was no correlation either between the p53 mutation and down-regulation of MHC class I alleles. The results suggest that in these tumours the mutated p53 is not the source of immunogenic peptides and that the lack of recognition of the tumours with mp53 is not caused by a defect in the expression of MHC class I molecules.     

<Emphasis Type="Italic">K-ras,BRCA1/2</Emphasis>, and <Emphasis Type="Italic">CHEK2</Emphasis> mutations and loss of heterozygosity at 9p, 17p,and 18q in sporadic adenocarcinoma of the pancreas

The diagnostic significance of molecular markers was assessed for the most common somatic aberrations at the K-ras, TP53, CDKN2A, and MADH4 loci, as well as less common mutations of BRCA1, BRCA2, and CHEK2, arising in preinvasive stages of sporadic adenocarcinoma of the pancreas. The study was performed on paired primary pancreatic adenocarcinoma and normal pancreatic tissue specimens obtained from 37 Russian patients. Surgical adenocarcinoma specimens were subjected to manual microdissection. Mutations of K-ras codon 12 were found in 24 tumor specimens (0.65), but not in normal pancreatic tissue specimens. Mutations of BRCA1 (185delAG, 300T > G, 4153delA, 4158A > G, 5382insC), BRCA2 (695insT, 6174delT), and CHEK2 (1100delC) were not found. The informativeness of allelic losses did not differ significantly among the three tumor suppressor loci and was 60% for TP53 (GDB186817) and CDKN2A (D9S974 + D9S162) and 65.7% for MADH4 (D18S363 + D18S474) (t = 0.48). The CDKN2A locus had the highest LOH frequency of 0.95. For TP53 and MADH4 the LOH frequency was 0.62 and 0.70, respectively. In 80% of adenocarcinomas, at least one locus was characterized with LOH. The overall informativeness of the combined data on K-ras mutations and loss of heterozygosity at 9p, 17p, and 18q was 85.7%. Only 9% of the tumors were characterized with microsatellite instability.     

Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease.

Epithelial cancers inflict a heavy human and social burden. It was estimated [Tyczynski JE, Bray F, Parkin DM. Lung cancer in Europe in 2000: epidemiology, prevention, and early detection. Lancet Oncol 2003;4:45-55 (Review)] that in Europe, in the year 2000, 347 000 persons died of lung cancer alone, the deadliest cancer disease. Loss of heterozygosity and large homozygous deletions of the human chromosome region 3p21 are especially frequent in epithelial cancers of several organs. In fact, 3p21 is a very peculiar region of the genome harbouring, tightly clustered, several types of cancer-causing genes (CCG) (Lerman MI, Minna JD. The 630 kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumour suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 2000;60:6116-33). Current results show that, unlike it was thought initially, many tumour suppressor genes (TSG) are located close by even in small genomic regions. They may be involved, perhaps with varying role, in different types of tumour, and may be influenced by the genetic background of different human populations as well as by environmental pollutants (cigarette smoking, professional exposure). This review will discuss methods of molecular analysis of genomic deletions to uncover CCG at 3p21, will summarize the present knowledge regarding the TSGs located in this region, and will describe a possible model of epithelial cancer pathogenesis.     

Identification of the amino acids in the Major Histocompatibility Complex class II region of Scottish Blackface sheep that are associated with resistance to nematode infection

Lambs with the Major Histocompatibility Complex DRB1*1101 allele have been shown to produce fewer nematode eggs following natural and deliberate infection. These sheep also possess fewer adult Teladorsagia circumcincta than sheep with alternative alleles at the DRB1 locus. However, it is unclear if this allele is responsible for the reduced egg counts or merely acts as a marker for a linked gene. This study defined the MHC haplotypes in a population of naturally infected Scottish Blackface sheep by PCR amplification and sequencing, and examined the associations between MHC haplotypes and faecal egg counts by generalised linear mixed modelling. The DRB1*1101 allele occurred predominately on one haplotype and a comparison of haplotypes indicated that the causal mutation or mutations occurred in or around this locus. Additional comparisons with another resistant haplotype indicated that mutations in or around the DQB2*GU191460 allele were also responsible for resistance to nematode infections. Further analyses identified six amino acid substitutions in the antigen binding site of DRB1*1101 that were significantly associated with reductions in the numbers of adult T. circumcincta.     

Linkage analysis of idiopathic generalized epilepsy (IGE) and marker loci on chromosome 6p in families of patients with juvenile myoclonic epilepsy: no evidence for an epilepsy locus in the HLA region.

Evidence for a locus (EJM1) in the HLA region of chromosome 6p predisposing to idiopathic generalized epilepsy (IGE) in the families of patients with juvenile myoclonic epilepsy (JME) has been obtained in two previous studies of separately ascertained groups of kindreds. Linkage analysis has been undertaken in a third set of 25 families including a patient with JME and at least one first-degree relative with IGE. Family members were typed for eight polymorphic loci on chromosome 6p: F13A, D6S89, D6S109, D6S105, D6S10, C4B, DQA1/A2, and TCTE1. Pairwise and multipoint linkage analysis was carried out assuming autosomal dominant and autosomal recessive inheritance and age-dependent high or low penetrance. No significant evidence in favor of linkage was obtained at any locus. Multipoint linkage analysis generated significant exclusion data (lod score < -2.0) at HLA and for a region 10-30 cM telomeric to HLA, the extent of which varied with the level of penetrance assumed. These observations indicate that genetic heterogeneity exists within this epilepsy phenotype.     

Exclusive KRAS mutation in microsatellite-unstable human colorectal carcinomas with sequence alterations in the DNA mismatch repair gene, MLH1

Microsatellite instability (MSI) is regarded as reflecting defective DNA mismatch repair (MMR). MMR defects lead to an increase in point mutations, as well as repeat instability, on the genome. However, despite the highly unstable microsatellites, base substitutions in representative oncogenes or tumor suppressors are extremely infrequent in MSI-positive tumors. Recently, the heterogeneity in MSI-positive colorectal tumors is pointed out, and the 'hereditary' and 'sporadic settings' are proposed. Particularly in the former, base substitution mutations in KRAS are regarded as relatively frequent. We sequenced the KRAS gene in a panel of 76 human colorectal carcinomas in which the MSI status has been determined. KRAS mutations were detected in 22 tumors (28.9%). Intriguingly, all of the KRAS-mutant MSI-H (high) tumors harbored sequence alterations in an essential MMR gene, MLH1, which implies that KRAS mutation more frequently and almost exclusively occurs in MMR gene-mutant MSI-H tumors. Furthermore, in contrast with the prevailing viewpoint, some of these tumors are derived from sporadic colorectal cancer patients. The tight connection between MMR gene mutation and KRAS mutation may suggest previously unrecognized complexities in the relationship between MSI and the mutator phenotype derived from defective MMR.