Burkhard H. A. von Rahden

Common variants in the HLA-DQ region confer susceptibility to idiopathic achalasia

Idiopathic achalasia is characterized by a failure of the lower esophageal sphincter to relax due to a loss of neurons in the myenteric plexus. This ultimately leads to massive dilatation and an irreversibly impaired megaesophagus. We performed a genetic association study in 1,068 achalasia cases and 4,242 controls and fine-mapped a strong MHC association signal by imputing classical HLA haplotypes and amino acid polymorphisms. An eight-residue insertion at position 227–234 in the cytoplasmic tail of HLA-DQβ1 (encoded by HLA-DQB1*05:03 and HLA-DQB1*06:01) confers the strongest risk for achalasia (P = 1.73 × 10−19). In addition, two amino acid substitutions in the extracellular domain of HLA-DQα1 at position 41 (lysine encoded by HLA-DQA1*01:03; P = 5.60 × 10−10) and of HLA-DQβ1 at position 45 (glutamic acid encoded by HLA-DQB1*03:01 and HLA-DQB1*03:04; P = 1.20 × 10−9) independently confer achalasia risk. Our study implies that immune-mediated processes are involved in the pathophysiology of achalasia.

LgR5 expression and cancer stem cell hypothesis: clue to define the true origin of esophageal adenocarcinomas with and without Barrett's Esophagus?

BackgroundInvestigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barretts Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis.Materials and methodsExpression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates.ResultsLgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNA-level, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis.ConclusionThe stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.

MMP-1 is a (pre-)invasive factor in Barrett-associated esophageal adenocarcinomas and is associated with positive lymph node status

BackgroundEsophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barretts esophagus (BE) regarded as precancerous lesion. Matrix metalloproteinases (MMPs) might play a role during the multistep carcinogenetic process.MethodsExpression of MMP-1 and -13 was analyzed in esophageal cancer (n = 41 EAC with BE, n = 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC), furthermore in BE without intraepithelial neoplasia (IN) (n = 18), and the cell line OE-33. MMP-1 was co-labelled with Ki-67 (proliferation), Cdx-2 (marker for intestinal metaplasia, BE) and analyzed on mRNA level. MMP-1 staining results were correlated with clinicopatholocical parameters.ResultsOn protein level, MMP-1 expression was found in 39 of 41 (95%) EAC with BE, in 19 of 19 (100%) EAC without BE, in 6 of 10 (60%) ESCC, and in 10 of 18 (56%) BE without IN. No expression of MMP-13 was found in these specimens. Quantification showed 48% MMP-1 positive cells in EAC with BE, compared to 35% in adjacent BE (p < 0.05), 44% in EAC without BE, 32% in ESCC, and 4% in BE without IN. Immunofluorescence double staining experiments revealed increased MMP-1 expressing in proliferating cells (MMP-1+/Ki-67+) (r = 0.943 for BE and r = 0.811 for EAC). On mRNA-level, expression of MMP-1 was significantly higher in EAC compared to BE (p = 0.01) and confirmed immunohistochemical staining results. High MMP-1 levels were associated with lymph node metastases but not with poorer survival (p = 0.307).ConclusionsOur findings suggest that MMP-1 plays a role as preinvasive factor in BE-associated EAC. Expression of MMP-1 in proliferating BE and EAC cells suggest malignant proliferation following the clonal expansion model.

Tumour-mediated TRAIL-Receptor expression indicates effective apoptotic depletion of infiltrating CD8+ immune cells in clinical colorectal cancer

Expression of apoptosis-related proteins on tumour cells has been shown in several experimental models to be an efficient mechanism for a counterattack against host anti-tumour immune responses in solid tumours. Here we provide a clinical evidence for such a tumour immune escape mechanism by demonstrating tumour to T cell-directed death receptor signalling (TRAIL/TRAIL-Receptor (TRAIL-R)) in colorectal cancer (CRC). In a series of patients with CRC and completed 5-year follow up, we investigated apoptosis and expression levels of apoptosis-related proteins. Gene and protein profiles in the tumours demonstrated intratumoural upregulated gene expression for Fas, Fas-L, TRAIL, TRAIL-R and TNF-alpha (RT-qPCR). Levels of terminaldeoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labelling (TUNEL)-positive events were positively correlated with TRAIL-R1-expression on tumour infiltrating immune cells. Among the immune cells, preferentially CD8+ T cells were found to express TRAIL-R1 while serial immunostaining in the same patient tumours showed abundant apoptotic (TUNEL-positive) immune cells. In conclusion, our results in tumour samples from CRC patients suggest TRAIL-R1-mediated apoptotic depletion of infiltrating immune cells (CD8+) in response to TRAIL expression by the tumour itself. This supports the notion of an efficient escape from tumour immune response and thus evasion from the attack of activated CD8+ T cells. These findings may enhance our understanding of tumour progression in CRC and might be helpful for the development of TRAIL and its death receptor-based therapy.

Comprehensive epidemiological and genotype–phenotype analyses in a large European sample with idiopathic achalasia

Background and aim Although an eight-residue insertion in HLA-DQ&bgr;1 has been recently identified as a genetic risk factor for idiopathic achalasia, other risk factors are still unknown. In the present study, we carried out an epidemiological survey and a genotype–phenotype (G×P) analysis to gain further insights into the etiology of achalasia. Methods We obtained medical data from 696 achalasia patients and 410 controls, as well as their first-degree relatives (2543 of patients and 1497 of controls). For the G×P analysis, we stratified the patients into HLA-DQ&bgr;1 insertion carriers and noncarriers. Results Our data show that patients are more often affected by viral infections before achalasia onset (P<0.0001, most significantly for varicella zoster virus infections). In addition, allergic (P=0.0005) and autoimmune disorders (P=0.0007, most significantly for psoriasis and Sjögren’s syndrome) represent comorbid disease conditions. First-degree relatives of patients also show higher prevalence rates of allergic disorders (P=0.0007) and psoriasis (P=0.016) compared with control relatives. Moreover, the G×P analysis reveals that achalasia is triggered by pregnancies in female HLA-DQ&bgr;1 insertion carriers (P=0.031). Conclusion Our data point to a role of viral infections in the development of achalasia. In addition, they provide evidence for a relationship between achalasia and allergic, as well as autoimmune, disorders. Furthermore, pregnancy seems to be a disease-triggering factor in female HLA-DQ&bgr;1 insertion carriers, which points to hormonal and/or immunosuppressive factors influencing disease development.

Ten-year follow-up of a prospective trial for the targeted therapy of gastric cancer with the human monoclonal antibody PAT-SC1

The fully human monoclonal antibody PAT-SC1 is specific for an isoform of CD55 (decay-accelerating factor) designated CD55PAT-SC1. This antigen is expressed in the majority (80%) of gastric cancers (GCs), and the antibody induces tumour cell-specific apoptosis in vitro as well as in vivo. PAT-SC1, therefore, has been deemed promising as a therapeutic agent. Here, we describe the results of an academic clinical study performed in a neoadjuvant setting with resectable GC patients. Patients undergoing treatment for GC between 1997 and 2001 were tested for CD55PAT-SC1 expression. Fifty-one resectable patients that tested positively received a single administration of 20 mg PAT-SC1 48 h prior to surgery. They underwent standard surgery with either subtotal or total gastrectomy with bursectomy, omentectomy and a modified D2-lymphadenectomy, aimed at R0 resection. Primary endpoints of the present study were to evaluate side-effects of the PAT-SC1 antibody treatment and to evaluate histopathological effects such as tumour regression and induction of apoptosis. Long-term survival was a secondary endpoint. Administration of PAT-SC1 appeared safe with only reversible side-effects according to WHO grade I and II. Despite the low-dose of the antibody, 81.6% of the patients showed signs of increased apoptosis within the primary tumour and 60% showed signs of tumour cell regression. Comparison of the 10-year survival rates of the R0-resected CD55PAT-SC1-positive patients treated with the PAT-SC1 antibody with a historical collective of R0-resected CD55PAT-SC1-positive patients not treated with PAT-SC1 indicated a survival benefit in the treated patients. Furthermore, comparison of the patient survival of CD55PAT-SC1-positive vs. CD55PAT-SC1-negative groups suggested that CD55PAT-SC1 antigen expression is an independent predictor of poor survival in a Cox regression analysis. Antibody PAT-SC1 may be a useful additive therapeutic agent in the treatment of patients with CD55PAT-SC1-expressing GCs. In combination with radical standard surgery, PAT-SC1 given as an adjuvant or neoadjuvant immunotherapeutic agent induces apoptosis in tumour cells which may improve survival of these patients. Because of the human origin and its specific binding to the CD55PAT-SC1 antigen, PAT-SC1 was well tolerated in this trial.

The HLA-DQβ1 insertion is a strong achalasia risk factor and displays a geospatial north–south gradient among Europeans

Idiopathic achalasia is a severe motility disorder of the esophagus and is characterized by a failure of the lower esophageal sphincter to relax due to a loss of neurons in the myenteric plexus. Most recently, we identified an eight-amino-acid insertion in the cytoplasmic tail of HLA-DQβ1 as strong achalasia risk factor in a sample set from Central Europe, Italy and Spain. Here, we tested whether the HLA-DQβ1 insertion also confers achalasia risk in the Polish and Swedish population. We could replicate the initial findings and the insertion shows strong achalasia association in both samples (Poland P=1.84 × 10−04, Sweden P=7.44 × 10−05). Combining all five European data sets – Central Europe, Italy, Spain, Poland and Sweden – the insertion is achalasia associated with Pcombined=1.67 × 10−35. In addition, we observe that the frequency of the insertion shows a geospatial north–south gradient. The insertion is less common in northern (around 6–7% in patients and 2% in controls from Sweden and Poland) compared with southern Europeans (~16% in patients and 8% in controls from Italy) and shows a stronger attributable risk in the southern European population. Our study provides evidence that the prevalence of achalasia may differ between populations.