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A letter to the editor is presented related to mixed chimerism in allogeneic stem cell transplantation.

The PvuII (rs2234693) Single Nucleotide Polymorphism (SNP) in the gene coding for the estrogen receptor-1 (ESR1), has been found associated with outcome in tamoxifen treated patients with early hormone-receptor positive breast cancer. However, it remains unclear whether this SNP is a predictive marker for tamoxifen efficacy or a prognostic marker for breast cancer outcome. The aim of this study was to examine the prognostic potential of this SNP in postmenopausal early breast cancer patients treated with adjuvant exemestane. Dutch postmenopausal patients randomised to 5 years of adjuvant exemestane of whom tissue was available (N = 807) were selected from the Tamoxifen Exemestane Adjuvant Multinational (TEAM) trial database. The SNP rs2234693 in the ESR1 gene was genotyped on DNA from formalin-fixed paraffin embedded (FFPE) tumor tissue using Taqman assays and related to the primary endpoint disease-free survival (DFS) and secondary endpoint overall survival (OS). Survival analyses were performed using Cox regression analysis. In total 805 patients were included in the analyses (median follow up of 5.22 years) and genotypes were obtained in 97% of the samples. The variant T allele of PvuII in ESR1 (rs2234693) was associated with a better DFS (hazard ratio (HR) 0.689, 95% confidence interval (CI) 0.480-0.989, P = 0.044) in univariate analysis only, and a better OS in both univariate (HR 0.616, 95%, CI 0.411-0.923, P = 0.019) and multivariate analyses (HR 0.571, 95% CI 0.380-0.856, P = 0.007), consistent with a prognostic rather than a predictive drug response effect. Variation of PvuII in the ESR1 gene is related to OS in postmenopausal, early HR + breast cancer patients treated with exemestane in the TEAM study. Variation in the ESR1 gene may therefore be a prognostic marker of early breast cancer survival, and warrants further research.

Despite the nationwide availability of pharmacogenomic (PGx) guidelines in electronic medication surveillance systems in The Netherlands, PGx guided prescribing is still uncommon in primary care. We set out to investigate the adoption of pharmacist initiated PGx testing in primary care. Community pharmacists were offered a free PGx test covering 40 variants in 8 genes to test patients receiving an incident prescription (IRx) of a selection of 10 drugs. Results of the PGx test along with predicted phenotypes and a therapeutic recommendation based on the Dutch Pharmacogenetics Working Group (DPWG) guidelines were transferred to the pharmacist and physician. Adoption was defined as the percentage of eligible patients that received genotyping. From November 2014-July 2016, 200 patients were included with an adoption of 18.0%. Of the included patients 57.5% received an IRx for atorvastatin, 14.5% started with simvastatin and 28.0% received an IRx for amitriptyline, (es)citalopram, nortriptyline, or venlafaxine. 90% of the patients carried at least one actionable PGx test result in the selected PGx-panel. In 31.0% of the incident prescriptions a combination between a drug with a known gene-drug interaction and an actionable genotype was present and a therapeutic recommendation was provided. The provided recommendations were accepted by the clinicians in 88.7% of the patients. Pharmacist initiated implementation of PGx in primary care is feasible, and the frequency of actionable gene-drug interactions for the selected drugs is high.

Aim: The aim of our study was to explore the potential of FcGR genetic polymorphisms as a predictor of adalimumab efficacy in rheumatoid arthritis (RA) patients., Materials & Methods: The study population was composed of 302 Dutch RA patients receiving adalimumab therapy. The FcGR2A (R131>H; rs1801274) and FcGR3A (F158>V; rs396991) genetic variants were genotyped using the TaqMan(®) allelic discrimination technology. Treatment outcome was evaluated with the use of the 28-joint disease activity score criteria (DAS28) and good response and remission were classified according to European League Against Rheumatism (EULAR) criteria., Results: Comparing allelic frequencies between responders and nonresponders, the presence of the FcGR2A*R allele was associated with EULAR good response at 14 weeks (p = 0.017, odds ratio: 1.53, 95% CI: 1.08-2.17). No significant association was found for FcGR3A, with good response or remission. The combined effect of both FcGR2A and FcGR3A SNPs showed a trend for association with EULAR good response (p-value = 0.041, odds ratio: 1.38, 95% CI: 1.01-1.89)., Conclusion: Our results indicate that FcGR polymorphisms could be a determinant of adalimumab efficacy in RA patients. Original submitted 28 July 2014; Revision submitted 19 December 2014.

Monoclonal antibodies, such as rituximab, trastuzumab, and cetuximab, mediate immune response by binding to Fcγ receptors. The frequently occurring Phe158Val variant of the FCGR3A gene has increased binding affinity and consequently may affect immune response. Several pharmacogenetic association studies have genotyped this variant (FCGR3A rs396991), but with disconcordant results. In addition, in some of these studies genotype distribution was not in Hardy-Weinberg equilibrium, and samples were excluded from analysis because of genotype inconsistency. Genotyping problems of FCGR3A rs396991 are most likely due to sequence homology with the FCGR3B gene. For that reason, we developed a novel pyrosequencing method specifically for genotyping FCGR3A rs396991 and confirmed that the FCGR3B gene is not coamplified.

Purpose: In a recent randomized phase III clinical trial in metastatic colorectal cancer patients, the addition of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab to bevacizumab and chemotherapy resulted in decreased progression-free survival, in particular for patients with the high-affinity FcγRIIIA., Experimental Design: The presence of natural killer (NK) cells and type 2 (M2) macrophages in colorectal cancer was determined by immunohistochemistry, using antibodies to lineage-specific markers NKp46 and CD68 with CD163, respectively. Influence of tumor-bound cetuximab on M2 macrophages was carried out in vitro with EGFR-expressing tumor cells and short-term differentiated monocytes from blood donors, who were typed for the FcγRIIIA polymorphism (CD16)., Results: Antibody-dependent cellular cytotoxicity by NK cells is generally proposed as one of the antitumor mechanisms of mAbs. We found that CD163-positive M2 macrophages are much more abundant in colorectal carcinomas. In vitro analysis of M2 macrophages revealed high levels of Fc-gamma receptors (FcγR) and PD-L1 and production of IL-10 and VEGF but not IL-12. These anti-inflammatory and tumor-promoting mediators were released upon coculture with EGFR-positive tumor cells loaded with low concentrations of cetuximab. Macrophage activation depended on EGFR expression on the tumor cells, FcγRs, target specificity of the mAb and mobility of antibody complexes. Cetuximab-induced macrophage responses were more pronounced for FCGR3A 158-Val (high-affinity) carriers., Conclusion: These results suggest that tumor-promoting M2 macrophages are activated by the therapeutic mAb cetuximab in the local tumor microenvironment and argue that this immune mechanism should be taken into account for the application of therapeutic antibodies., (©2011 AACR.)

DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is often fragmented and cross-linked and is therefore difficult to genotype. To enable this source of DNA for genotyping analysis using Taqman probes, we tested whether enrichment of the target genes would increase the amount of available DNA. For enrichment of the target genes, we used preamplification by means of diluted Taqman assays. To establish the appropriateness of preamplification, we used DNA extracted from paraffin-embedded tissue and compared the genotyping results of a series of single nucleotide polymorphisms assessed in DNA samples with and without preamplification. In a subset of patients, DNA was isolated from both blood and FFPE tissue to test the reliability of genotyping results derived after preamplification. We found an increase in call rate after preamplification and a convincing concordance in genotype. Based on our findings, we can safely conclude that preamplification of DNA isolated from paraffin-embedded tissue is a valuable and reliable method to optimize genotyping results.

Objectives: Genetic variation in genes encoding for drug-metabolizing enzymes, drug targets and signaling pathways have proven to contribute significantly to differences in drug response. Pharmacogenetics is now expanding from clinical pharmacological research to its application in clinical practice. Genotyping of patients in a routine clinical setting requires robust and reliable genotyping methods., Materials & Methods: A survey of pharmacogenetic association studies for quality control samples published from 2005 to 2007 in the two most prominent pharmacogenetic journals, and development of plasmid-derived external controls., Results: Surveying journals revealed that only a minority of papers report the use of quality controls, and no standard procedures are applied. We established 12 plasmid-derived external controls and applied these in pharmacogenetic testing., Conclusion: There still is a need for quality control materials, especially for application in pharmacogenetic testing. We hope that our initiative to create plasmid-derived controls will help to facilitate quality in the pharmacogenetic genotyping tests applied in research, as well as in routine patient care.

Objective: Recombinant human GH (rhGH) replacement in adults is aimed at improving signs and symptoms of the adult GH deficiency (GHD) syndrome. In children, a common polymorphism of the GH receptor (exon-3 deletion, d3GHR) increases the response to rhGH replacement. The aim of this study was to assess the effects of this polymorphism on the response to rhGH replacement in adults., Design: Prospective intervention with rhGH during 1 yr (n = 99) and in a subset during 5 yr (n = 53)., Patients and Methods: The presence of the d3GHR variant was established in GHD patients and linked to short-term and long-term effects of rhGH replacement on IGF-I, lipid metabolism, anthropometric parameters, and bone mineral density., Results: Fifty-five patients had two wild-type alleles (56%), whereas 38 patients (38%) had one allele and six patients (6%) had two alleles coding the d3GHR isoform. During short-term rhGH replacement, the increase in IGF-I was higher in patients bearing at least one d3GHR allele, compared with those with two wild-type alleles (at an identical mean dose of rhGH). The decrease in total cholesterol and low-density lipoprotein cholesterol was lower in the group bearing at least one d3GHR allele, whereas the increase in high-density lipoprotein cholesterol was higher, compared with patients with the wild-type genotype. In contrast, these differential responses of GHR genotype could not be demonstrated during long-term rhGH replacement., Conclusion: The d3GHR genotype contributes, at least for some parameters, to the interindividual differences in efficacy of short-term, but not long-term, rhGH replacement in adults with GHD.

Upon infection with mycobacteria the IL-12/IFN-gamma axis plays an essential role in the activation of cell-mediated immunity required for the elimination of pathogens. Mutations in genes of the IL-12/IFN-gamma axis are known to cause extreme susceptibility to infection with environmental mycobacteria, and subtle variations in these genes may influence susceptibility to more virulent mycobacteria. We analyzed the distribution of polymorphisms in four essential genes from the IL-12/IFN-gamma axis, IL12B, IL12RB1, IFNG and IFNGR1, in 382 pulmonary tuberculosis patients and 437 healthy controls from an endemic region in Jakarta, Indonesia. The IL12RB1 gene was sequenced in a subset of individuals. Nine known single nucleotide polymorphisms (SNPs) and two new silent variations, 135G>A and 1056C>T, were detected in IL12RB1. Six functional SNPs (-2C>T, 467G>A, 641A>G, 1312C>T, 1573G>A, 1781G>A) in IL12RB1, an IL12B promoter insertion/deletion polymorphism and CA repeats in IFNG and IFNGR1 were analyzed in the cohort. The IFNGR1 allele CA(12) (p=0.004) and genotype CA(12)/CA(12) (p=0.01; OR 0.5) were associated with protection from pulmonary tuberculosis. Interestingly, IL12B promoter heterozygosity was associated with protection from tuberculosis in BCG-vaccinated individuals (p=0.03; OR=0.6). This new finding supports the role that IL-23-of which IL12B encodes a subunit--plays in generation of memory T cells.

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.