Specific T-cell receptor beta-rearrangements of gluten-triggered CD8+ T-cells are enriched in celiac disease patients' duodenal mucosa.

Celiac disease (CeD) is an autoimmune disorder affecting the small intestine with gluten as disease trigger. Infections including Influenza A, increase the CeD risk. While gluten-specific CD4+ T-cells, recognizing HLA-DQ2/DQ8 presented gluten-peptides, initiate and sustain the celiac immune response, CD8+ α/ß intraepithelial T-cells elicit mucosal damage. Here, we subjected TCRs from a cohort of 56 CeD patients and 22 controls to an analysis employing 749 published CeD-related TCRß-rearrangements derived from gluten-specific CD4+ T-cells and gluten-triggered peripheral blood CD8+ T-cells. We show, that in addition to TCRs from gluten-specific CD4+ T-cells, TCRs of gluten-triggered CD8+ T-cells are significantly enriched in CeD duodenal tissue samples. TCRß-rearrangements of gluten-triggered CD8+ T-cells were even more expanded in patients than TCRs from gluten-specific CD4+ T-cells (p < 0.0002) and highest in refractory CeD. Sequence alignments with TCR-antigen databases suggest that a subgroup of these most likely indirectly gluten-triggered TCRs recognize microbial, viral, and autoantigens.

Prognostic impact of activin subunit inhibin beta A in gastric and esophageal adenocarcinomas.

PURPOSE: Adenocarcinomas of the esophagus (AEG) and stomach (AS) are among the most common cancers worldwide. Novel markers for risk stratification and guiding treatment are strongly needed. Activin is a multi-functional cytokine with context specific pro- and anti-tumorigenic effects. We aimed to investigate the prognostic role of activin tumor protein expression in AEG/ASs. METHODS: Tissue from a retrospective cohort of 277 patients with AEG/AS treated primarily by surgery at the Charité - Universitätsmedizin Berlin was collected and analyzed by immunohistochemistry using a specific antibody to the activin homodimer inhibin beta A. Additionally, we evaluated T-cell infiltration and PD1 expression as well as expression of PD-L1 by immunohistochemistry as possible confounding factors. Clinico-pathologic data were collected and correlated with activin protein expression. RESULTS: Out of 277 tumor samples, 72 (26.0%) exhibited high activin subunit inhibin beta A protein expression. Higher expression was correlated with lower Union for International Cancer Control (UICC) stage and longer overall survival. Interestingly, activin subunit expression correlated with CD4+ T-cell infiltration, and the correlation with higher overall survival was exclusively seen in tumors with high CD4+ T-cell infiltration, pointing towards a role of activin in the tumor immune response in AEG/ASs. CONCLUSION: In our cohort of AEG/AS, higher activin subunit levels were correlated with longer overall survival, an effect exclusively seen in tumors with high CD4+ cell infiltration. Further mechanistic research is warranted discerning the exact effect of this context specific cytokine.

A genetically defined signature of responsiveness to erlotinib in early-stage pancreatic cancer patients: Results from the CONKO-005 trial.

BACKGROUND: high recurrence rates of up to 75% within 2 years in pancreatic ductal adenocarcinoma (PDAC) patients resected for cure indicate a high medical need for clinical prediction tools and patient specific treatment approaches. Addition of the EGFR inhibitor erlotinib to adjuvant chemotherapy failed to improve outcome but its efficacy in some patients warrants predictors of responsiveness. PATIENTS AND METHODS: we analysed tumour samples from 293 R0-resected patients from the randomized, multicentre phase III CONKO-005 trial (gemcitabine ± erlotinib) with targeted sequencing, copy number, and RNA expression analyses. FINDINGS: a total of 1086 mutations and 4157 copy-number aberrations (CNAs) with a mean of 17.9 /tumour were identified. Main pathways affected by genetic aberrations were the MAPK-pathway (99%), cell cycle control (92%), TGFß signalling (77%), chromatin remodelling (71%), and the PI3K/AKT pathway (65%). Based on genetic signatures extracted with non-negative matrix factorization we could define five patient clusters, which differed in mutation patterns, gene expression profiles, and survival. In multivariable Cox regression analysis, SMAD4 aberrations were identified as a negative prognostic marker in the gemcitabine arm, an effect that was counteracted when treated with erlotinib (DFS: HR=1.59, p = 0.016, and OS: HR = 1.67, p = 0.014). Integration of differential gene expression analysis established SMAD4 alterations with low MAPK9 expression (n = 91) as a predictive biomarker for longer DFS (HR=0.49; test for interaction, p = 0.02) and OS (HR = 0.32; test for interaction, p = 0.001). INTERPRETATION: this study identified five biologically distinct patient clusters with different actionable lesions and unravelled a previously unappreciated association of SMAD4 alteration status with erlotinib effectiveness. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that SMAD4 status might guide addition of erlotinib treatment in early-stage PDAC patients.

Better prognosis of gastric cancer patients with high levels of tumor infiltrating lymphocytes is counteracted by PD-1 expression.

The prognostic potential of anti-tumor immune responses is becoming increasingly important in adenocarcinoma of the gastroesophageal junction and stomach (AGE/S) especially regarding the use of immune checkpoint inhibitors. This study analyzes for the first time the prognostic impact of tumor-infiltrating lymphocytes (TILs) and checkpoint inhibitors in a large Caucasian cohort in patients with AGE/S. We screened tissue samples from 438 therapy-naïve patients with AGE/S undergoing surgery between 1992 and 2005, examined in a tissue microarray (TMA) and stained against human CD3, CD4, CD8, PD-1, and PD-L1. Out of 438 tissue samples, 210 were eligible for multivariate analysis. This revealed that high infiltration with CD3+, CD4+, or CD8+ TILs was associated with an increased overall survival in AGE/S patients, which could only be confirmed in multivariate analysis for CD3 (HR: 0.326; p = .023). Independent improved survival was limited to gastric cancer patients and to early tumor stages as long as TILs did not express PD-1 (HR: 1.522; p = .021). Subgroup analyses indicate that TIL-dependent anti-tumor immune response is only effective in gastric cancer patients in early stages of disease in PD-1 negative TILs. Combined analysis of PD-1 and CD3 could serve as a prognostic marker for the clinical outcome of gastric cancer patients and could also be of interest for immunotherapy.

Prognostic impact of Claudin 18.2 in gastric and esophageal adenocarcinomas.

INTRODUCTION: The tight junction molecule Claudin 18.2 is selectively expressed in healthy and malignant gastric epithelial tissue and is a promising therapy target for high Claudin 18.2 expressing adenocarcinomas of the esophagogastric junction and stomach (AEG/S). METHODS: This study analyzed the prevalence, characteristics and prognostic impact of Claudin 18.2 expression in primary tumor, lymph node and distant metastasis in a large Caucasian AGE/S cohort with 414 patients. RESULTS: Claudin 18.2 was highly expressed in 17.1% of primary tumors, 26.7% of lymph node metastasis and 16.7% of distant metastasis. High Claudin 18.2 expression in lymph node metastasis and primary tumors correlated significantly (p < 0.001). High expression of Claudin 18.2 was neither associated with histomorphogical subtype, or tumor state, nor with overall survival. CONCLUSION: In Caucasian AEG/S patients, 17.1% appeared to be eligible for an anti-Claudin 18.2 therapy. Claudin 18.2 expression itself has no impact on prognosis and is not related to any tumor subtype.

Evidence for a role of RUNX1 as recombinase cofactor for TCRß rearrangements and pathological deletions in ETV6-RUNX1 ALL.

T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

[Retrospective evaluation of relevance of care in the management of presumed benign ovarian tumors]. / Évaluation rétrospective de la pertinence des soins dans la prise en charge des tumeurs ovariennes présumées bénignes.

INTRODUCTION: The relevance of care is defined by the right act, for the right patient, at the right time. We were interested in the relevance of the management of presumed benign ovarian tumors before and after the release of the CNGOF guidelines 2013 (French guidelines). METHODS: This is a retrospective observational study conducted at the University Hospital in Strasburg France from 01/01/2013 to 31/12/2017 including all patients treated for a presumed benign ovarian cyst. We were interested in the diagnostic approach: relevance of the prescribed imaging and the use of CA 125 dosage, in the therapeutic approach: the relevance of the technique used as well as the relevance of the surgical indication. We compared our practices between 2013 and 2017 for these same items. RESULTS: We included 682 cysts for 621 patients, the imaging performed was relevant in 55% of cases, not relevant but justified in 25% and irrelevant in 20%. The CA 125 assay or its absence of assay was relevant in 84% of cases. The surgical technique was relevant in 67% of cases and not relevant but justified in 29%. With a significant improvement 7.1% in 2013 of irrelevant against 0.9% for the year 2017. The surgical indication was relevant in 72% of cases, not relevant but justified in 20% and irrelevant in 2.7%. CONCLUSIONS: The analysis of the relevance of care allows an evaluation of our practices. Professional recommendations can have an impact on the quality of care.

Comparative characteristics of older people with type 1 diabetes treated with continuous subcutaneous insulin infusion or insulin injection therapy: data from the German/Austrian DPV registry.

AIM: To compare clinical characteristics and outcomes in adults with type 1 diabetes aged ≥ 60 years using continuous subcutaneous insulin infusion (CSII) vs. insulin injection therapy. Further, to determine the percentage of older adults with type 1 diabetes using CSII. RESEARCH DESIGN AND METHODS: Retrospective study using data of the Diabetes Prospective Follow-up Registry (DPV). Including percentage CSII use from 2008 to 2018, and the characteristics of 9547 individuals extracted from the DPV in March 2019 (N = 1404 CSII; N = 8143 insulin injection therapy). Wilcoxon rank sum tests were used for continuous variables and chi-square tests for categorical variables to compare clinical characteristics of people using CSII vs. insulin injection therapy. Adjusted analyses used generalized linear models to compare diabetes-related outcomes. RESULTS: CSII usage has increased in older adults (from 12% in 2008 to 23% in 2018). After adjustment, CSII was associated with lower HbA1c [60.7 mmol/mol (7.7 ± 0.1%) vs. 62.8% (7.9 ± 0.1%)], lower daily insulin dose (0.49 ± 0.02 vs. 0.61 ± 0.01 IU/kg), fewer days in hospital (8.1 ± 0.12 vs. 11.2 ± 0.11 days/person-year), fewer severe hypoglycaemic events (0.16 ± 0.02 vs. 0.21 ± 0.03 events/person-year) and fewer diabetic ketoacidosis (0.06 ± 0.01 vs. 0.08 ± 0.01 events/person-year). Individuals on CSII showed lower rates of microalbuminuria and also have a diagnosis of depression and neuropathy. CONCLUSIONS: A growing number of older adults are using insulin pumps. Older age in itself should not be seen as a contraindication for CSII.

Analysis of NTRK expression in gastric and esophageal adenocarcinoma (AGE) with pan-TRK immunohistochemistry.

Small molecule inhibitors such as Larotrectinib have been recently approved in the treatment of patients with fusions of the neurotrophic-tropomyosin receptor tyrosine kinase (NTRK) genes 1-3. These genomic rearrangements have been reported across different tumor subtypes with a high prevalence in rare tumors. However, in gastric and esophageal adenocarcinoma (AGE) NTRK fusions have also been described in a subset of Asian patients. In order to study the prevalence of this alteration in Caucasian patients with AGE we performed immunohistochemistry for pan-NTRK in 438 formalin-fixed paraffin embedded (FFPE) tumor samples. While we found NTRK expression in gastric glands and tumor adjacent nerve tissue, we did not detect this marker in the tumor compartment. Based on our findings NTRK fusions do not seem to play a role in the molecularpathology of Caucasian AEG patients, so that other treatment options are required.

Interference of tumour mutational burden with outcome of patients with head and neck cancer treated with definitive chemoradiation: a multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group.

BACKGROUND: Tumour mutational burden (TMB) estimated from whole exome sequencing or comprehensive gene panels has previously been established as predictive factor of response to immune checkpoint inhibitors (ICIs). Its predictive value for the efficacy of concurrent chemoradiation (cCRTX), a potential combination partner of ICI, remains unknown. METHODS: The accuracy of TMB estimation by an in-house 327-gene panel was established in the Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) data set. Interference of TMB with outcome after cCRTX was determined in a multicentre cohort of patients with locally advanced HNSCC uniformly treated with cCRTX. Targeted next-generation sequencing was successfully applied in 101 formalin-fixed, paraffin-embedded pretreatment tumour samples. In a subset of cases (n = 40), tumour RNA was used for immune-related gene expression profiling by the nanoString platform. TMB was correlated with TP53 genotype, human papilloma virus (HPV) status, immune expression signatures and survival parameters. Results were validated in the TCGA HNSCC cohort. RESULTS: A high accuracy of TMB estimation by the 327-gene panel was established. High TMB was significantly associated with an increased prevalence of TP53 mutations and immune gene expression patterns unrelated to T cell-inflamed gene expression profiles. Kaplan-Meier analysis revealed significantly reduced overall survival in the patient group with high TMB (hazard ratio for death: 1.79, 95% confidence interval: 1.02-3.14; P = 0.042) which remained significant after correcting for confounding factors in the multivariate model. The prognostic value of TMB was confirmed in the TCGA HNSCC cohort. CONCLUSION: High TMB identifies HNSCC patients with poor outcome after cCRTX who might preferentially benefit from CRTX-ICI combinations.

[The construction and operation of a central biomaterial bank : The ZeBanC of the Charité Berlin]. / Der Aufbau und Betrieb einer Zentralen Biomaterialbank : Die ZeBanC der Charité Berlin.

The use of high-quality, well-characterized biomaterials and their clinical and demographic information for biomedical research has steadily increased in importance over the last 10 years. There are several reasons for this. On the one hand, several studies have shown that even in high-profile scientific papers the description of the origin and characteristics of the biomaterials used is missing or insufficient. On the other hand, it has become generally accepted that small collections of biomaterials are often created under ethically and legally unclear conditions without quality measures and without sufficient sustainability.The establishment of a centralized biomaterial bank (ZeBanC) was initiated in order to steer the biomaterial collections at the Charité under well defined conditions. This activity is carried out in close cooperation between the Charité and the Berlin Institute of Health (BIH).

Hysteroscopic enlargement metroplasty for T-shaped uterus: 24 years' experience at the Strasbourg Medico-Surgical and Obstetrical Centre (CMCO).

STUDY QUESTION: What is the impact of hysteroscopic enlargement metroplasty for T-shaped uterus on the live birth rate? SUMMARY ANSWER: Performing enlargement metroplasty appears to improve the obstetrical prognosis and fertility in patients with a T-shaped uterus. WHAT IS KNOWN ALREADY: T-shaped uterus is linked to an excess of myometrium in the uterine walls giving rise to a subcornual constriction ring which causes dysmorphism and hypoplasia of the uterine cavity. It is commonly associated with infertility or a sequence of repeated miscarriages. STUDY DESIGN: Single-centre observational cohort study in 112 patients who underwent enlargement metroplasty for T-shaped uterus between 1992 and 2016 in a Strasbourg university hospital centre. MAIN RESULTS: The mean age of patients was 33.2; they had been attempting to conceive on average for 56 months for subfertile patients and 42.2 months for infertile patients. Prior to surgery, patients had succeeded in becoming pregnant 161 times, i.e. a mean gravidity of 1.4 pregnancies. For subfertile patients the mean gravidity was 2.67. Mean parity was 0.04. In the overall population, one hundred pregnancies occurred following enlargement metroplasty. The live birth rate increased in a statistically significant manner following enlargement metroplasty: 4 (2.5%) vs. 60 (60%), p < 0.05. In parallel, the miscarriage rate was statistically reduced: 126 (78.3%) vs. 22 (22%), pnull< .05. Intraoperative complications were 1 case of cervical laceration (0.9%) and 1 case of false passage (0.9%). Subsequent pregnancies remained at risk of miscarriage (22%) and premature delivery (20%) but not extra uterine gestation. Delivery took place by Caesarean section in 61% of cases. In the subgroup of infertile patients, the live birth rate was also markedly increased and 49% of pregnancies which occurred were spontaneous. LIMITATIONS: This study was descriptive and retrospective. WIDER IMPLICATIONS: These results are consistent with those in the literature. Hysteroscopic enlargement metroplasty is now a well-established technique with few complications but which should nevertheless be reserved for symptomatic patients.

[Requirements for a cross-location biobank IT infrastructure : Survey of stakeholder input on the establishment of a biobank network of the German Biobank Alliance (GBA)]. / Anforderungen an eine standortübergreifende Biobanken-IT-Infrastruktur : Erhebung des Stakeholderinputs zum Aufbau eines Biobankennetzwerks der German Biobank Alliance (GBA).

BACKGROUND: The large number of biobanks within Germany results in a high degree of heterogeneity with regard to the IT components used at the respective locations. Within the German Biobank Alliance (GBA), 13 biobanks implement harmonized processes for the provision of biomaterial and accompanying data. OBJECTIVES: The networking of the individual biobanks and the associated harmonisation of the IT infrastructure should facilitate access to biomaterial and related clinical data. METHODS: For this purpose, the relevant target groups were first identified in order to determine their requirements for IT solutions to be developed in a workshop. RESULTS: Of the seven identified interest groups, three were initially invited to a first round of discussions. The stakeholder input expressed resulted in a catalogue of requirements with regard to IT support for (i) a sample and data request, (ii) the handling of patient consent and inclusion, and (iii) the subsequent evaluation of the sample and data request. CONCLUSIONS: The next step is to design the IT solutions as prototypes based on these requirements. In parallel, further user groups are being surveyed in order to be able to further concretise the specifications for development.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Differences between BCL2-break positive and negative follicular lymphoma unraveled by whole-exome sequencing.

Depending on disease stage follicular lymphoma (FL) lack the t(14;18) in ~15-~50% of cases. Nevertheless, most of these cases express BCL2. To elucidate mechanisms triggering BCL2 expression and promoting pathogenesis in t(14;18)-negative FL, exonic single-nucleotide variant (SNV) profiles of 28 t(14;18)-positive and 13 t(14;18)-negative FL were analyzed, followed by the integration of copy-number changes, copy-neutral LOH and published gene-expression data as well as the assessment of immunoglobulin N-glycosylation sites. Typical FL mutations also affected t(14;18)-negative FL. Curated gene set/pathway annotation of genes mutated in either t(14;18)-positive or t(14;18)-negative FL revealed a strong enrichment of same or similar gene sets but also a more prominent or exclusive enrichment of immune response and N-glycosylation signatures in t(14;18)-negative FL. Mutated genes showed high BCL2 association in both subgroups. Among the genes mutated in t(14;18)-negative FL 555 were affected by copy-number alterations and/or copy-neutral LOH and 96 were differently expressed between t(14;18)-positive and t(14;18)-negative FL (P<0.01). N-glycosylation sites were detected considerably less frequently in t(14;18)-negative FL. These results suggest a diverse portfolio of genetic alterations that may induce or regulate BCL2 expression or promote pathogenesis of t(14;18)-negative FL as well as a less specific but increased crosstalk with the microenvironment that may compensate for the lack of N-glycosylation.