Treatment outcome of atypical EGFR mutations in the German National Network Genomic Medicine Lung Cancer (nNGM).

BACKGROUND: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. PATIENTS AND METHODS: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). RESULTS: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. CONCLUSIONS: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.

Distribution of tumor-infiltrating-T-lymphocytes and possible tumor-escape mechanisms avoiding immune cell attack in locally advanced adenocarcinomas of the esophagus

Introduction The inflammatory microenvironment has emerged as one of the focuses of cancer research. Little is known about the immune environment in esophageal adenocarcinoma (EAC) and possible tumor-escape mechanisms to avoid immune cell attack. Patients and methods We measured T cell inflammation (CD3, CD8) in the microenvironment using a standardized software-based evaluation algorithm considering different predefined tumor areas as well as expression of MHC class 1 and PD-L1 on 75 analyzable primarily resected and locally advanced (≥ pT2) EACs. We correlated these findings statistically with clinical data. Results Patients with high amounts of T cell infiltration in their tumor center showed a significant survival benefit of 41.4 months compared to 16.3 months in T cell poor tumors (p = 0.025), although CD3 fails to serve as an independent prognostic marker in multivariate analysis. For the invasion zone, a correlation between number of T-cells and overall survival was not detectable. Loss of MHC1 protein expression on tumor cells was seen in 32% and PD-L1 expression using the combined positive score (CPS) in 21.2%. Most likely due to small numbers of cases, both markers are not prognostically relevant, even though PD-L1 expression correlates with advanced tumor stages. Discussion Our analyses reveal an outstanding, though not statistically independent, prognostic relevance of T-cell-rich inflammation in our group of EACs, in particular driven by the tumor center. For the first time, we describe that the inner part of the invasion zone in EACs shows significantly fewer T-cells than other tumor segments and is prognostically irrelevant. We also demonstrate that the loss of antigen presenting ability via MHC1 downregulation by the carcinoma cells is a common escape mechanism in EACs. Future work will need to show whether tumors with MHC class 1 loss respond less well to immunotherapy (AU)

Distribution of tumor-infiltrating-T-lymphocytes and possible tumor-escape mechanisms avoiding immune cell attack in locally advanced adenocarcinomas of the esophagus.

INTRODUCTION: The inflammatory microenvironment has emerged as one of the focuses of cancer research. Little is known about the immune environment in esophageal adenocarcinoma (EAC) and possible tumor-escape mechanisms to avoid immune cell attack. PATIENTS AND METHODS: We measured T cell inflammation (CD3, CD8) in the microenvironment using a standardized software-based evaluation algorithm considering different predefined tumor areas as well as expression of MHC class 1 and PD-L1 on 75 analyzable primarily resected and locally advanced (≥ pT2) EACs. We correlated these findings statistically with clinical data. RESULTS: Patients with high amounts of T cell infiltration in their tumor center showed a significant survival benefit of 41.4 months compared to 16.3 months in T cell poor tumors (p = 0.025), although CD3 fails to serve as an independent prognostic marker in multivariate analysis. For the invasion zone, a correlation between number of T-cells and overall survival was not detectable. Loss of MHC1 protein expression on tumor cells was seen in 32% and PD-L1 expression using the combined positive score (CPS) in 21.2%. Most likely due to small numbers of cases, both markers are not prognostically relevant, even though PD-L1 expression correlates with advanced tumor stages. DISCUSSION: Our analyses reveal an outstanding, though not statistically independent, prognostic relevance of T-cell-rich inflammation in our group of EACs, in particular driven by the tumor center. For the first time, we describe that the inner part of the invasion zone in EACs shows significantly fewer T-cells than other tumor segments and is prognostically irrelevant. We also demonstrate that the loss of antigen presenting ability via MHC1 downregulation by the carcinoma cells is a common escape mechanism in EACs. Future work will need to show whether tumors with MHC class 1 loss respond less well to immunotherapy.

In vivo imaging of translocator protein, a marker of activated microglia, in alcohol dependence.

Neuroinflammation may be a critical component of the neurobiology of alcohol use disorders, yet the exact nature of this relationship is not well understood. This work compared the brain and peripheral immune profile of alcohol-dependent subjects and controls. Brain levels of 18-kDa translocator protein (TSPO), a marker of microglial activation and neuroinflammation, were measured with [11C]PBR28 positron emission tomography imaging in 15 healthy controls and 15 alcohol-dependent subjects. Alcohol-dependent subjects were imaged 1-4 days (n=14) or 24 days (n=1) after their last drink. Linear mixed modeling of partial-volume-corrected [11C]PBR28 data revealed a main effect of alcohol dependence (P=0.034), corresponding to 10% lower TSPO levels in alcohol-dependent subjects. Within this group, exploratory analyses found a negative association of TSPO levels in the hippocampus and striatum with alcohol dependence severity (P<0.035). Peripheral immune response was assessed in a subset of subjects by measuring cytokine expression from monocytes cultured both in the presence and absence of lipopolysaccharide. Peripheral monocyte response to lipopolysaccharide stimulation was lower in alcohol-dependent subjects compared with controls for the proinflammatory cytokines interleukin-6 and interleukin-8. Thus, alcohol-dependent individuals exhibited less activated microglia in the brain and a blunted peripheral proinflammatory response compared with controls. These findings suggest a role for pharmaceuticals tuning the neuroimmune system as therapeutics for alcohol dependence.

Imaging of cerebral α4ß2* nicotinic acetylcholine receptors with (-)-[(18)F]Flubatine PET: Implementation of bolus plus constant infusion and sensitivity to acetylcholine in human brain.

The positron emission tomography (PET) radioligand (-)-[(18)F]flubatine is specific to α4ß2(⁎) nicotinic acetylcholine receptors (nAChRs) and has promise for future investigation of the acetylcholine system in neuropathologies such as Alzheimer's disease, schizophrenia, and substance use disorders. The two goals of this work were to develop a simplified method for α4ß2(⁎) nAChR quantification with bolus plus constant infusion (B/I) (-)-[(18)F]flubatine administration, and to assess the radioligand's sensitivity to acetylcholine fluctuations in humans. Healthy human subjects were imaged following either bolus injection (n=8) or B/I (n=4) administration of (-)-[(18)F]flubatine. The metabolite-corrected input function in arterial blood was measured. Free-fraction corrected distribution volumes (VT/fP) were estimated with modeling and graphical analysis techniques. Next, sensitivity to acetylcholine was assessed in two ways: 1. A bolus injection paradigm with two scans (n=6), baseline (scan 1) and physostigmine challenge (scan 2; 1.5mg over 60min beginning 5min prior to radiotracer injection); 2. A single scan B/I paradigm (n=7) lasting up to 240min with 1.5mg physostigmine administered over 60min beginning at 125min of radiotracer infusion. Changes in VT/fP were measured. Baseline VT/fP values were 33.8±3.3mL/cm(3) in thalamus, 12.9±1.6mL/cm(3) in cerebellum, and ranged from 9.8 to 12.5mL/cm(3) in other gray matter regions. The B/I paradigm with equilibrium analysis at 120min yielded comparable VT/fP values with compartment modeling analysis of bolus data in extrathalamic gray matter regions (regional means <4% different). Changes in VT/fP following physostigmine administration were small and most pronounced in cortical regions, ranging from 0.8 to 4.6% in the two-scan paradigm and 2.8 to 6.5% with the B/I paradigm. These results demonstrate the use of B/I administration for accurate quantification of (-)-[(18)F]flubatine VT/fP in 120min, and suggest possible sensitivity of (-)-[(18)F]flubatine binding to physostigmine-induced changes in acetylcholine levels.

Evidence for a polygenic contribution to androgenetic alopecia.

BACKGROUND: Male pattern baldness (androgenetic alopecia, AGA) is a highly heritable trait and the most common form of hair loss in humans. Eight genome-wide significant risk loci for AGA have been identified. OBJECTIVES: To determine whether a polygenic component contributes to the genetic risk for AGA. METHODS: This study used a German case-control sample for AGA, which comprised 581 severely affected patients and 617 controls, to determine the contribution of polygenic variance to AGA risk. The sample was divided evenly into discovery and test samples. An additive polygenic risk score was calculated from risk alleles with increasingly liberal P-values in the discovery dataset, which was then used to test for the enrichment of AGA risk score alleles in the independent test samples. RESULTS: The polygenic score analysis provided significant evidence for a polygenic contribution to AGA where the amount of variance explained was 1·4-4·5%. CONCLUSION: This study provides evidence for the specific contribution of a polygenic component to the overall heritable risk for AGA. To some degree, the polygenic architecture of AGA might reflect the complexity of the biological pathways involved. Further analyses and strategies that complement conventional genome-wide association studies are needed to identify these factors. These may include pathway-based analyses, the analysis of functional candidate genes and tests for epistatic effects with known loci.

Susceptibility variants on chromosome 7p21.1 suggest HDAC9 as a new candidate gene for male-pattern baldness.

BACKGROUND: Male-pattern baldness (androgenetic alopecia, AGA) is the most common form of hair loss among humans. Research has shown that it is caused by genetic factors. Numerous studies have unequivocally identified two major genetic risk loci for AGA: the X-chromosomal AR/EDA2R locus, and the PAX1/FOXA2 locus on chromosome 20. OBJECTIVES: To identify further candidate genes for AGA, and thus gain further insights into this phenotype. METHODS: A German sample of 581 severely affected cases and 617 controls was used to perform a genome-wide association study. The identified associated locus was further analysed by fine-mapping, and then independently replicated in an Australian sample. Expression and pathway analyses were performed to characterize the susceptibility gene identified. RESULTS: The most significant association signal was obtained for rs756853 (P = 1·64 × 10(-7) ), which is located intronically in the histone deacetylase 9 (HDAC9) gene. Fine-mapping and a family-based analysis revealed that rs756853 and the 6-kb distal rs2249817 were the most highly associated single nucleotide polymorphisms. The association finding was replicated in an independent Australian sample, when the analysis was restricted to severely affected cases and unaffected controls (P = 0·026). Analysis of rs2249817 in a combined sample of severely affected German and Australian cases and unaffected controls revealed a strong association signal (P = 9·09 × 10(-8) ). Tissue expression studies demonstrated HDAC9 expression in various tissues, including tissues of relevance to AGA. No strong genotypic effects were observed in genotype-specific expression or splice studies. Pathway analyses supported the hypothesis that HDAC9 plays a functional role in AGA via interaction with the AR gene. CONCLUSIONS: The present study suggests that HDAC9 is the third AGA susceptibility gene.

Specific α4ß2 nicotinic acetylcholine receptor binding of [F-18]nifene in the rhesus monkey.

OBJECTIVE: [F-18]Nifene is a PET radioligand developed to image α4ß2* nicotinic acetylcholine receptors (nAChR) in the brain. This work assesses the in vivo binding and imaging characteristics of [F-18]nifene in rhesus monkeys for the development of PET experiments examining nAChR binding. METHODS: Dynamic PET imaging experiments with [F-18]nifene were acquired in four anesthetized Macaca mulatta (rhesus) monkeys using a microPET P4 scanner. Data acquisition was initiated with a bolus injection of 109 ± 17 MBq [F-18]nifene and the time course of the radioligand in the brain was measured for up to 120 min. For two experiments, a displacement dose of (-)nicotine (0.03 mg kg(-1) , i.v.) was given 45-60 min post injection and followed 30 min later with a second [F-18]nifene injection to measure radioligand nondisplaceable uptake. Time activity curves were extracted in the regions of the antereoventral thalamus (AVT), lateral geniculate nucleus region (LGN), frontal cortex, and the cerebellum (CB). RESULTS: The highest levels of [F-18]nifene uptake were observed in the AVT and LGN. Target-to-CB ratios reached maximum values of 3.3 ± 0.4 in the AVT and 3.2 ± 0.3 in the LGN 30-45 min postinjection. Significant binding of [F-18]nifene was observed in the subiculum, insula cortex, temporal cortex, cingulate gyrus, frontal cortex, striatum, and midbrain areas. The (-)nicotine displaced bound [F-18]nifene to near background levels within 15 min postdrug injection. No discernable displacement was observed in the CB, suggesting its potential as a reference region. Logan graphical estimates using the CB as a reference region yielded binding potentials of 1.6 ± 0.2 in the AVT and 1.3 ± 0.1 in the LGN. The postnicotine injection displayed uniform nondisplaceable uptake of [F-18]nifene throughout gray and white brain matter. CONCLUSIONS: [F-18]Nifene exhibits rapid equilibration and a moderately high target to background binding profile in the α4ß2* nAChR rich regions of the brain, thus providing favorable imaging characteristics as a PET radiotracer for nAChR assay.

In vivo kinetics of [F-18]MEFWAY: a comparison with [C-11]WAY100635 and [F-18]MPPF in the nonhuman primate.

UNLABELLED: [F-18]Mefway was developed to provide an F-18 labeled positron emission tomography (PET) neuroligand with high affinity for the serotonin 5-HT(1A) receptor to improve the in vivo assessment of the 5-HT(1A) system. The goal of this work was to compare the in vivo kinetics of [F-18]mefway, [F-18]MPPF, and [C-11]WAY100635 in the rhesus monkey. METHODS: Each of four monkeys were given bolus injections of [F-18]mefway, [C-11]WAY100635, and [F-18]MPPF and scans were acquired with a microPET P4 scanner. Arterial blood was sampled to assay parent compound throughout the time course of the PET experiment. Time activity curves were extracted in the high 5-HT(1A) binding areas of the anterior cingulate cortex (ACG), mesial temporal cortex, raphe nuclei, and insula cortex. Time activity curves were also extracted in the cerebellum, which was used as a reference region. The in vivo kinetics of the radiotracers were compared based on the nondisplaceable distribution volume (V(ND) ) and binding potential (BP(ND) ). RESULTS: At 30 min, the fraction of radioactivity in the plasma due to parent compound was 19%, 28%, and 29% and cleared from the arterial plasma at rates of 0.0031, 0.0078, and 0.0069 (min⁻¹) ([F-18]mefway, [F-18]MPPF, [C-11]WAY100635). The BP(ND) in the brain regions were mesial temporal cortex: 7.4 ± 0.6, 3.1 ± 0.4, 7.0 ± 1.2, ACG: 7.2 ± 1.2, 2.1 ± 0.2, 7.9 ± 1.2; raphe nuclei: 3.7 ± 0.6, 1.3 ± 0.3, 3.3 ± 0.7; and insula cortex: 4.2 ± 0.6, 1.2 ± 0.1, 4.7 ± 1.0 for [F-18]mefway, [F-18]MPPF, and [C-11]WAY100635 respectively. CONCLUSIONS: In the rhesus monkey, [F-18]mefway has similar in vivo kinetics to [C-11]WAY100635 and yields greater than 2-fold higher BP(ND) than [F-18]MPPF. These properties make [F-18]mefway a promising radiotracer for 5-HT(1A) assay, providing higher counting statistics and a greater dynamic range in BP(ND).

A genome-wide association study in 574 schizophrenia trios using DNA pooling.

The cost of genome-wide association (GWA) studies can be prohibitively high when large samples are genotyped. We conducted a GWA study on schizophrenia (SZ) and to reduce the cost, we used DNA pooling. We used a parent-offspring trios design to avoid the potential problems of population stratification. We constructed pools from 605 unaffected controls, 574 SZ patients and a third pool from all the parents of the patients. We hybridized each pool eight times on Illumina HumanHap550 arrays. We estimated the allele frequencies of each pool from the averaged intensities of the arrays. The significance level of results in the trios sample was estimated on the basis of the allele frequencies in cases and non-transmitted pseudocontrols, taking into account the technical variability of the data. We selected the highest ranked SNPs for individual genotyping, after excluding poorly performing SNPs and those that showed a trend in the opposite direction in the control pool. We genotyped 63 SNPs in 574 trios and analysed the results with the transmission disequilibrium test. Forty of those were significant at P<0.05, with the best result at P=1.2 x 10(-6) for rs11064768. This SNP is within the gene CCDC60, a coiled-coil domain gene. The third best SNP (P=0.00016) is rs893703, within RBP1, a candidate gene for schizophrenia.

The R620W polymorphism in PTPN22 confers general susceptibility for the development of alopecia areata.

BACKGROUND: The functional R620W (c.1858C>T) variant of the protein tyrosine phosphatase nonreceptor 22 gene (PTPN22) has been associated with a variety of autoimmune disorders. A recent study has suggested that R620W also contributes to the severe form of alopecia areata (AA). OBJECTIVES: We sought to replicate the finding of an association between PTPN22 and severe AA. In addition, we wanted to study the effect of PTPN22 on the general risk to develop AA and on other subtypes of AA (mild AA, early/late age at onset, positive/negative family history). METHODS: The R620W variant was genotyped in a large case-control sample of Belgian-German origin with 435 patients and 628 controls. RESULTS: Significant results were obtained for the overall collective of patients with AA (P=0.007). Subdividing the sample according to severity of AA, family history and age at onset, we detected lowest P-values for patients with the severe form of AA (Pcorr=0.036), with a positive family history (Pcorr=0.042) and with an age at onset

The hairless gene in androgenetic alopecia: results of a systematic mutation screening and a family-based association approach.

BACKGROUND: Genetic disposition and androgen dependence are important characteristics of the common patterned loss of scalp hair known as androgenetic alopecia (AGA). The genetic factors contributing to AGA are currently unknown. The human hairless gene (HR) has recently been cloned and mutations have been reported in families with autosomal recessive universal congenital alopecia and papular atrichia. The main feature of these disorders is persistent complete absence of hair at or shortly after birth. This suggests that HR is essential and specific for the development of hair. OBJECTIVES: To test the hypothesis that HR may be involved in AGA. METHODS: We systematically screened HR for genetic variability by means of single-strand conformation analysis (SSCA) in 46 unrelated men with AGA. To test for an involvement of HR in the development of AGA, seven common variants were genotyped in 61 families with 93 affected offspring. The results were analysed with the transmission/disequilibrium test (TDT). RESULTS: SSCA showed 15 single nucleotide substitutions: eight missense mutations, four silent mutations and three mutations in exon-flanking intronic sequences. TDT results showed a marginally significant association between AGA and variants 3379-29G/T (P = 0.024) and 2611-68C/T (P = 0.047). These results, however, did not remain significant after applying the conservative Bonferroni correction for multiple testing. CONCLUSIONS: Our results do not provide evidence for a strong involvement of HR in the development of AGA, although a minor role cannot be fully excluded.

A distinct gene close to the hairless locus on chromosome 8p underlies hereditary Marie Unna type hypotrichosis in a German family.

BACKGROUND: Hypotrichosis of the Marie Unna type (HMU) is a rare autosomal dominant disorder characterized by male-pattern hair loss with childhood onset and anomalies of the hair shaft. OBJECTIVES: We aimed to evaluate a number of chromosomal loci as possible candidate regions for HMU. METHODS: A linkage analysis was performed in a large German family using microsatellite markers spanning candidate regions on chromosomes 8, 12 and 17. RESULTS: We found that the HMU locus maps to chromosomal region 8p21 in a 13.01-cM interval between markers D8S1145 and D8S1771. This interval harbours the hairless gene (HR). Mutational analysis of HR on the genomic and transcript levels revealed no pathogenic mutation. CONCLUSIONS: Our findings, together with a recent report of two unrelated families of Dutch and British origin, provide evidence for a hair growth regulatory gene distinct from HR in chromosomal region 8p21.

Novel Hairless mutations in two kindreds with autosomal recessive papular atrichia.

Papular atrichia is an autosomal recessive disorder characterized clinically by the occurrence of universal congenital alopecia and disseminated papular lesions. Recently, mutations in the human hairless (HR) gene have been reported in Irish and Arab Palestinian families with papular atrichia. We have studied two further kindreds with this clinical phenotype from other ethnic backgrounds. For mutation detection the complete coding region as well as exon-intron boundaries of the HR gene were sequenced. The first family is a Mexican family with clinically typical papular atrichia. Sequencing identified a homozygous deletion of 4 bp in exon 7 (2001delCCAG) leading to a premature stop codon in exon 8. The second family is a South Tyrolian family with affected individuals showing papular atrichia and retardation of bone age during childhood. All affected individuals were identified as homozygous for an A-->G transition at nucleotide position 2909 (exon 14) leading to an amino acid change of asparagine to serine in codon 970 (Asn970Ser). These data provide further evidence for the involvement of hairless mutations in papular atrichia. In addition, these findings suggest that the hairless protein is not only involved in hair development but also in the process of ossification during development.

Cloning, genomic organization, alternative transcripts and mutational analysis of the gene responsible for autosomal recessive universal congenital alopecia.

Complete or partial congenital absence of hair (congenital alopecia) may occur isolated or with associated defects. The majority of families with isolated congenital alopecia has been reported to follow an autosomal recessive mode of inheritance (MIM 203655). We have previously mapped the gene for autosomal recessive congenital alopecia in a large inbred Pakistani family in which affected persons show complete absence of hair development (universal congenital alopecia) to a 15 cM region on chromosome 8p21-22. Here we report the cloning and characterization of the human homologue of the mouse hairless gene and show that it is located in the critical region on chromosome 8p21-22. Determining the exon-intron structure allowed detailed mutational analysis of DNA samples of patients with universal congenital alopecia. We detected a homozygous missense mutation in the Pakistani family and a homozygous splice donor mutation in a family from Oman. In addition, we show that the human hairless gene undergoes alternative splicing and that at least two isoforms generated by alternative usage of exon 17 are found in human tissues. Interestingly, the isoform containing exon 17 is the predominantly expressed isoform in all tissues but skin, where exclusive expression of the shorter isoform was observed. We speculate that this tissue-specific difference in the proportion of hairless transcripts lacking exon 17 sequences could contribute to the tissue-specific disease phenotype observed in individuals with isolated congenital alopecia.