Features of a new full length HLA allele: A*02:455.

The full length sequence of HLA-A*02:455 differs from HLA-A*02:01:01:01 by one amino acid substitution: A245E.

Peptide-induced HLA-E expression in human PBMCs is dependent on peptide sequence and the HLA-E genotype.

Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.

RNA and protein expression of HLA-A(∗)23:19Q.

The assignment of null alleles is clinically relevant in stem cell transplantation, in particular for donor selection. It is unclear how questionable (Q) alleles, having an unknown expression profile, should be considered in matching criteria. In this study we analyzed the RNA and protein expression profile of a questionable allele encountered in a sample of the Guadeloupe population: GD23Q, HLA-A(∗)23:19Q, 29:02:01. Full-length DNA sequencing of HLA-A(∗)23:19Q revealed a single polymorphism at position 619 (G>A) compared to HLA-A(∗)23:01:01. Serological typing showed only the presence of HLA-A29; HLA-A(∗)23:19Q was not detected on the cell surface. The absence of HLA-A(∗)23:19Q surface expression was shown by flow cytometry using a directly labeled monoclonal antibody and a panel of five indirectly labeled polyclonal antibodies all directed against HLA-A23 (HLA-A9) molecules. Allele specific amplification revealed the absence of intact full-length mRNA, but the presence of two major alternatively spliced mRNAs: sequencing identified that in one variant exon 3 is missing and in the other variant introns 2 and 3 are retained. Based upon the lack of HLA-A(∗)23:19Q surface expression and the presence of aberrant mRNA transcripts only, this study shows that HLA-A(∗)23:19Q is non-expressed.

The full length genomic sequence of a novel HLA-C*03 allele: HLA-C*03:219.

HLA-C*03:219 differs from HLA-C*03:04:01:01 by a non-synonymous substitution in the α3 domain (T216I).

Clinical and immunological significance of HLA-E in stem cell transplantation and cancer.

Human leukocyte antigen-E (HLA-E) is a nonclassical HLA class I molecule that canonically binds peptides derived from the leader sequence of classical HLA class I. HLA-E can also bind peptides from stress protein [e.g. heat shock protein 60 (Hsp60)] and pathogens, illustrating the importance of HLA-E for anti-viral and anti-tumor immunity. Like classical HLA class I molecules, HLA-E is ubiquitously expressed, however, it is characterized by only a very limited sequence variability and two dominant protein forms have been described (HLA-E*01:01 and HLA-E*01:03). HLA-E influences both the innate and the adaptive arms of the immune system by the engagement of inhibitory (e.g. NKG2A) and activating receptors [e.g. αß T cell receptor (αßTCR) or NKG2C] on NK cells and CD8 T cells. The effects of HLA-E on the cellular immune response are therefore complex and not completely understood yet. Here, we aim to provide an overview of the immunological and clinical relevance of HLA-E and HLA-E polymorphism in stem cell transplantation and in cancer. We review novel insights in the mechanism via which HLA-E expression levels are controlled and how the cellular immune response in transplantation and cancer is influenced by HLA-E.

HLA frequencies and associations in cystic fibrosis.

Cystic fibrosis (CF) is classically attributed to the dysfunction of the single CF transmembrane conductance regulator gene. The incidence of human leukocyte antigen (HLA) polymorphisms in different CF-associated diseases raises the question of an unequal distribution of HLA genotypes in CF. This study aimed to evaluate HLA gene frequencies and possible associations in CF patients compared with a control population. Frequencies of HLA-DRB1, HLA-DQA1 and HLA-DQB1, performed by intermediate resolution typing using Luminex sequence-specific oligonucleotide, and epitope counts were similar in 340 CF patients when compared with 400 control subjects. In conclusion, HLA-DRB1, -DQA1 and -DQB1 do not seem to influence susceptibility to CF. Whether HLA plays a role in the severity of CF disease needs to be investigated.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation.

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

Full-length HLA-DPB1 diversity in multiple alleles of individuals from Caucasian, Black, or Oriental origin.

Despite DP antigens have been shown to be stimulators of the mixed lymphocyte reaction, human leukocyte antigen-DPB1 is not considered in the matching criteria for hematopoietic stem cell transplantation (HSCT). The role of DPB1 matching in HSCT remains inconclusive because of contradictory findings in different studies. The concept of permissible and non-permissible mismatches might clarify these contradictory results. Although several groups have attempted to identify immunogenic epitopes in exon 2 to establish permissive and non-permissive allele groups, the direct correlation between individual exon 2 amino acids and epitopes with DPB1 immunogenicity is still not evident. We hypothesize that polymorphism within the entire molecule, including polymorphic variability in different ethnic groups, is crucial to unravel the function of DPB1 polymorphism. Using an RNA-based approach, we sequenced all frequent and available non-frequent DPB1 alleles full length from 148 samples representing 28 different DPB1 alleles from either Black, Caucasian, or Oriental origin. We identified various DPB1 alleles with, in addition to the exon 2 polymorphism, polymorphisms in exons 1, 3, 4, and 5. Based on this polymorphism outside exon 2, we defined one new allele. Two alleles with identical exon 2 polymorphism but differing outside exon 2 were identified in individuals of different ethnic groups. As T cell binding is not restricted to the polymorphic groove and polymorphism in the ß2 domain of the DP molecule affects CD4 interaction, full-length polymorphism should be considered to determine immunogenicity. Eventually, this knowledge will provide new insights in the classification of DPB1 polymorphism and more importantly will add new perspectives to the concept of permissiveness in transplantation.

Gastric adenocarcinoma in a 13-year-old boy: a diagnosis not often seen in this age group.

Gastric adenocarcinoma is not uncommon in the adult population, but in the pediatric population it is an extremely rare entity. A 13-year-old boy was referred to a pediatric oncology unit for evaluation of a tumor in the upper abdomen. Further investigation revealed an advanced stage gastric carcinoma with metastases suggestive for a hereditary cause. Awareness for uncommon diagnoses is a key issue in regard of accurate treatment and overall prognosis.

A high-throughput Taqman approach for the discrimination of HLA-E alleles.

An association between human leukocyte antigen (HLA) molecules and the incidence of various diseases has been recognized for years. Molecules of the HLA system also exert an important role on the clinical outcome after transplantation such as stem cell transplantation. The conventional HLA typing method is sequence based typing (SBT) which is reliable but laborious. The goal of this study was to develop a high-throughput Taqman assay to screen large panels for HLA-E alleles. Two functional HLA-E alleles, *0101 and *0103, have been identified. We set up the Taqman assay with genomic DNA as template to discriminate the three HLA-E genotypes: homozygous HLA-E*0101, heterozygous *0101, *0103 and homozygous *0103. This Taqman approach was validated by the comparison of results obtained with the typing results acquired by sequenced base typing (SBT). Additional screening of a large panel showed the pronounced discriminative capacity of the Taqman assay for HLA-E allele typing. The Taqman assay is a fast, reliable and consistent HLA-E allele typing method, especially useful to screen large panels.

NK-KIR ligand identification: a quick Q-PCR approach for HLA-C epitope typing.

Interaction of donor natural killer (NK)-cell-associated killer cell immunoglobulin-like receptors (KIRs) with the patient's human leukocyte antigen-C (HLA-C) ligands can result in an alloreactive NK response after haematopoietic stem cell transplantation. In many retrospective studies, additional HLA-C-typing data are required to predict NK-cell alloreactivity. We developed a Taqman assay using the quantitative polymerase chain reaction (Q-PCR) technique that facilitates HLA-C epitope typing, allowing the assignment of HLA-C group 1 or 2 alleles based on the dimorphism at residues 77 and 80 rather than based on the sequence specific priming (SSP) and sequence-based typing allele types. Q-PCR analysis for HLA-C epitope detection showed three clusters reflecting homozygous group 1 or 2 and heterozygous samples. This new approach introduces a quick HLA-C epitope screening method to define the presence of the ligand for the KIR-HLA-C interaction.

Chimerism analysis within 6 months of allogeneic stem cell transplantation predicts relapse in acute myeloid leukemia.

The role of chimerism analysis as a prognostic indicator of relapse after hematopoietic stem cell transplantation (SCT) is controversial. We monitored chimerism status by short tandem repeat-based polymerase chain reaction (PCR) in T- and non-T-cell subsets and retrospectively evaluated clinical outcome in 96 patients with acute myeloid leukemia after myeloablative (MA) or reduced-intensity conditioning SCT. Fifty-six percent of 80 patients in the MA group demonstrated complete donor chimerism (CC) at all time points, whereas 6% had decreasing mixed chimerism (MC), 8% stable MC, 25% increasing MC and 3% increasing and decreasing MC. In 16 RIC patients, these percentages were 12, 50, 6, 6 and 19, respectively, together with 6% nonengraftment. Forty-three out of 96 patients experienced relapse. The last chimerism evaluation before relapse revealed increasing MC in only eight patients. In samples taken between 1 and 6 months post SCT, CC/decreasing MC was significantly related with a lower risk of relapse (31 versus 83%, P<0.000) and mortality (38 versus 83%, P<0.000) than with MC/increasing MC. However, the development of relapse was very rapid. Only very frequent monitoring of chimerism status by highly sensitive methods might identify impending relapse and allow early immunological intervention.

High level of chromosome 15 aneuploidy in head and neck squamous cell carcinoma lesions identified by FISH analysis: limited value of beta2-microglobulin LOH analysis.

In cancer research, loss of heterozygosity (LOH), defined by microsatellite markers, is frequently used in the identification of gene loss. Especially, genomic alterations in the human leukocyte antigen (HLA) genes and the beta2-microglobulin (beta2m) gene on chromosome 15 are of interest regarding their function in the immune system. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in 11 head and neck squamous cell carcinoma (HNSCC) lesions using fluorescence in situ hybridization (FISH). The 11 tumors were selected out of 53 HNSCC lesions based upon beta2m LOH analysis and beta2m expression. Centromere 1 and 15 FISH were developed to determine the chromosome 15 copy number. Sequence-based mutation analysis of beta2m was conducted on tumors without beta2m expression; no mutations in the coding sequences were found. For five HNSCC lesions with LOH and beta2m expression, centromere 15 FISH indicated gain rather than loss. In the majority of the 11 HNSCC lesions, FISH showed centromere 1 and 15 heterogeneity throughout the tumor. Moreover, FISH indicated a more complex chromosome 1 and 15 distribution than could be concluded from microsatellite LOH analysis. Our results show that microsatellite LOH analysis does not represent the beta2m gene copy number and support the results obtained from comparative genomic hybridization (CGH) studies. Conclusions on genomic alterations in tumors cannot be based on LOH data only but depend on the results of immunohistochemical staining, FISH, and CGH.

Nonlethal transfusion associated graft-versus-host disease in a severe combined immunodeficient patient.

An X-linked severe combined immunodeficient (SCID) patient received a nonirradiated erythrocyte transfusion and developed transfusion-associated graft-versus-host disease (TAGVHD), which was controllable with high-dose corticosteroids. Haplo-identical SCT was performed, after a myeloablative conditioning regimen. At day +26, he developed GVHD. Chimerism studies revealed DNA of the erythrocyte transfusion donor (ETD) and recipient only. Because of early nonengraftment and the presence of alloreactive T cells of ETD origin, the patient was treated with an immunosuppressive conditioning regimen followed by a second SCT from the same donor. While tapering immunosuppression, he again developed mild GVHD, and DNA of ETD and bone marrow donor origin were both present. On cyclosporin, the ETD-DNA signal finally disappeared. High-resolution HLA typing revealed haplo-identity between BMD, ETD and the patient, which might have contributed to the relative mild course of the TAGVHD.

Prognostic factors in localised prostate cancer with emphasis on the application of molecular techniques.

Prostate cancer is the most prevalent malignancy in males in the Western world and the second leading cause of male cancer death. Prostate specific antigen (PSA) based screening and case finding leads to identification of early stage prostate cancer. It is often difficult to discriminate between patients that need curative treatment and those that can be managed conservatively. Prognostic factors are used to make this clinical decision. Based on the classification proposed by the American College of Pathologists and the World Health Organisation, selected prognostic factors in prostate cancer are described. Clinical applicable factors are stage, grade and serum PSA. Prognostic factors that are not routinely used (for various reasons) are ploidy, histological type and cancer volume in needle biopsies. All other factors (including circulating tumour cells, angiogenesis, growth factors, proliferation rate, apoptosis, nuclear morphometry, neuroendocrine differentiation, loss of chromosomal regions, tumour suppresser genes and adhesion molecules) are promising as prognostic factor although currently their use in clinical decisions is not recommended. The role of these factors in prostate cancer growth and their predictive value are discussed. The rapid developments in molecular techniques allow assessment of structure or function of thousands of genes in a prostate biopsy sample. We expect that molecular characterisation of tumour material will become a clinically important tool to predict prognosis in patients with localised prostate cancer.

P53 tumor suppressor gene mutations in laryngeal cancer and in recurrent disease following radiation therapy.

In this study we performed p53 sequencing based mutation analysis in laryngeal cancers and matched recurrent disease following irradiation. The question is if irradiation affects the DNA and introduces or deletes mutations so that p53 cannot be used as a clonal marker anymore. P53 mutations were identified in fresh-frozen laryngectomy specimens with either primary laryngeal cancers, treated by surgery and irradiation post-operative with local failure during follow-up, or with recurrent laryngeal cancers following primary irradiation. In 21 tumors the p53 status was analyzed by direct sequencing full-length mRNA through RT-PCR. DNA sequencing analysis of exons 2 through 11 was performed when RNA isolation could not be performed. The marker mutation identified in this way was detected by DNA sequencing of the corresponding exon in formalin-fixed deparaffinized tumor biopsy samples in respectively matched recurrent disease following surgery and irradiation or primary tumor before irradiation. DNA sequencing analysis of the corresponding exon of peripheral blood leukocytes excluded the presence of germline mutations or polymorphisms. In 16 out of 21 tumors (71%), a mutation was identified. Fifteen of these marker mutations were detected in the matched tumor biopsy sample (94%). The only case lacking the marker mutation probably was a second primary tumor. We conclude that we find no direct evidence for induction or loss of p53 mutations following irradiation. Consequently, p53 may be used as a diagnostic tool when histological examination fails, for example in discriminating between the presence of a second primary tumor in the same area versus recurrent disease.

Sensitive detection of p53 mutation: analysis by direct sequencing and multisequence analysis.

In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We encountered some differences between the previously published reference p53 sequence and all our sequences and identified polymorphism in six regions.