The lung microbiome in patients with pneumocystosis.

BACKROUND: Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal infection that is associated with a high morbidity and mortality in immunocompromised individuals. In this study, we analysed the microbiome of the lower respiratory tract from critically ill intensive care unit patients with and without pneumocystosis. METHODS: Broncho-alveolar fluids from 65 intubated and mechanically ventilated intensive care unit patients (34 PCP+ and 31 PCP- patients) were collected. Sequence analysis of bacterial 16S rRNA gene V3/V4 regions was performed to study the composition of the respiratory microbiome using the Illumina MiSeq platform. RESULTS: Differences in the microbial composition detected between PCP+ and PCP- patients were not statistically significant on class, order, family and genus level. In addition, alpha and beta diversity metrics did not reveal significant differences between PCP+ and PCP- patients. The composition of the lung microbiota was highly variable between PCP+ patients and comparable in its variety with the microbiota composition of the heterogeneous collective of PCP- patients. CONCLUSIONS: The lower respiratory tract microbiome in patients with pneumocystosis does not appear to be determined by a specific microbial composition or to be dominated by a single bacterial species.

[Immunotherapy of uveal melanoma: vaccination against cancer. Multicenter adjuvant phase 3 vaccination study using dendritic cells laden with tumor RNA for large newly diagnosed uveal melanoma]. / Immuntherapie beim Aderhautmelanom: Vakzination gegen Krebs. Multizentrische adjuvante Phase-III-Impfstudie mit Tumor-RNA-beladenen dendritischen Zellen bei neu diagnostizierten, großen Uveamelanomen.

Uveal melanomas are the most common malignant tumors of the eye. With modern molecular biological diagnostic methods, such as chromosome 3 typing and gene expression analysis, these tumors can be categorized into highly aggressive (monosomy 3, class II) and less aggressive forms. This molecular biological stratification is primarily important for determining the risk of these tumors as no therapy is currently available that is able to prevent or delay metastases. A randomized study of patients with a poor prognosis (monosomy 3) is currently being carried out in order to determine whether a cancer vaccine prepared from autologous (patient's own) dendritic cells and uveal melanoma RNA can prevent or delay progression and further metastases of this extremely aggressive form of cancer. Inclusion in the uveal melanoma study, which hopes to provide a potential therapeutic option for patients, is only possible if patients are referred to an institution that is able to manufacture and provide this vaccination before the patient is operated on or treated with radiation. Untreated tumor material is necessary for producing the vaccine on an individualized patient basis.

[Uveal melanoma: current insights into clinical relevance of genetic testing]. / Aktuelles zur klinischen Bedeutung genetischer Veränderungen bei malignen Melanomen der Uvea.

Uveal melanoma is the most common primary intraocular tumour in Caucasians. There are approximately 500 new cases of uveal melanoma in Germany per year and the incidence rate peaks at the age of 70 years. Half of all uveal melanoma patients develop metastatic disease, which can be observed even many years after successful treatment of the primary tumour. In most cases the liver is the location of first manifestation. Based on the chromosome 3 status uveal melanomas can be divided into two major classes that differ in their metastatic potential. Tumours with a high risk to metastasise usually show monosomy 3, whereas tumours showing disomy 3 rarely metastasise. If a patient wishes to know about his individual risk, prognostic testing of the primary tumour tissue can be performed after obtaining tumour material via transscleral or transretinal biopsy, or by enucleation. To date results of prognostic testing do not influence therapeutic strategies. Recently, major key genes involved in uveal melanoma development, GNAQ, GNA11, BAP1, SF3B1 and EIF1AX, have been identified. Mutation profiling, in addition to chromosomal 3 analysis, will further refine the classification or subclassification of uveal melanomas and will hopefully influence diagnostic or therapeutic concepts. Hereditary mutations in tumour suppressor gene BAP1 are associated with an increased risk for different tumour entities. Detection of germ line mutations in this tumour suppressor gene should implicate further general screening examinations of the patient to be able to detect these tumour entities. Moreover relatives of these patients should be offered a screening for BAP1 mutation.

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours.

BACKGROUND: Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations. METHODS: The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas. RESULTS: Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma. CONCLUSION: Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

Clinical and mutation data in 12 patients with the clinical diagnosis of Nager syndrome.

Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T > C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.

Molecular pathology of uveal melanoma.

Like other cancers, uveal melanomas (UM) are characterised by an uncontrolled, clonal, cellular proliferation, occurring as a result of numerous genetic, and epigenetic aberrations. Signalling pathways known to be disrupted in UM include: (1) the retinoblastoma pathway, probably as a result of cyclin D1 overexpression; p53 signalling, possibly as a consequence of MDM2 overexpression; and the P13K/AKT and mitogen-activated protein kinase/extracellular signal-related kinase pathway pathways that are disturbed as a result of PTEN and GNAQ/11 mutations, respectively. Characteristic chromosomal abnormalities are common and include 6p gain, associated with a good prognosis, as well as 1p loss, 3 loss, and 8q gain, which correlate with high mortality. These are identified by techniques such as fluorescence in situ hybridisation, comparative genomic hybridisation, microsatellite analysis, multiplex ligation-dependent probe amplification, and single-nucleotide polymorphisms. UM can also be categorised by their gene expression profiles as class 1 or class 2, the latter correlating with poor survival, as do BRCA1-associated protein-1 (BAP1) inactivating mutations. Genetic testing of UM has enhanced prognostication, especially when results are integrated with histological and clinical data. The identification of abnormal signalling pathways, genes and proteins in UM opens the way for target-based therapies, improving prospects for conserving vision and prolonging life.

Prognostic significance of chromosome 3 alterations determined by microsatellite analysis in uveal melanoma: a long-term follow-up study.

BACKGROUND: In uveal melanoma (UM), the most frequent primary intraocular tumour in adults, loss of one entire chromosome 3 (monosomy 3 (M3)) is observed in ~50% of tumours and is significantly associated with metastatic disease. The strong association of metastatic disease with M3 offers the opportunity for molecular prognostic testing of UM patients. METHODS: To re-evaluate M3 as prognostic marker in our clinical and laboratory setting and to determine the metastatic potential of rare tumours with partial M3, we performed a comprehensive study on 374 UM patients treated by enucleation in our clinic within 10 consecutive years, starting in 1998. Genotyping of all tumours was performed by microsatellite analysis. RESULTS: Median follow-up time was 5.2 years. The disease-specific mortality rates (death by UM metastases) for tumours with disomy 3 (D3) and M3 were 13.2% and 75.1%, respectively. The disease-specific survival was worse when M3 was observed together with chromosome 8 alterations (P=0.020). Death of UM metastases was also observed in 12 patients (9%) with D3 tumours. The metastasising D3 tumours showed a larger basal tumour diameter (P=0.007), and were more frequently of mixed or epitheloid cell type (P<0.0001) than D3 tumours that did not metastasise. Mortality rate of tumours showing partial M3 (8.3%) was as low as that for tumours with D3. CONCLUSION: This shows that large tumours with disomy 3 have an increased risk to develop metastases. On the basis of these results, our clinic offers routine prognostic testing of UM patients by chromosome 3 typing.

Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression.

Quantitative multiplex PCR and genomic real-time PCR were used to complete an RB1 mutation analysis in 57 of 433 and 72 of 262 patients with hereditary and isolated unilateral retinoblastoma, respectively. These patients were selected because in previous analyses, which focused mainly on the identification of point mutations, no RB1 mutation was found. We identified gross deletions and insertions in peripheral blood DNA from 26 of 57 patients (46%) with hereditary retinoblastoma, and in six of 72 patients (8.3%) with isolated unilateral disease. In addition, we identified 32 somatic mutations in tumor DNA from 31 of 72 patients (43%) with isolated unilateral retinoblastoma. Together with our previous results, we found that gross RB1 alterations were present in the peripheral blood DNA from 65 of 433 (15%) and 17 of 262 (6.5%) patients with bilateral or familial and isolated unilateral retinoblastoma, respectively. Including reported gross deletions, an analysis of the frequency of breakpoints per intron length shows higher densities in introns 13, 16, 23, and 24. Genotype-phenotype analyses showed that on the whole, carriers of gross deletions develop fewer retinoblastomas compared to patients who are heterozygous for other types of RB1 null mutations. Specifically, carriers of cytogenetic and submicroscopic whole gene deletions often have unilateral tumors only. By contrast, almost all patients with gross deletions with one breakpoint in RB1 have bilateral retinoblastoma.

[Prognostically relevant markers of malignant melanoma of the uvea]. / Prognostisch relevante Marker bei malignem Melanom der Uvea.

In addition to classic risk factors such as tumor size, tumor location, and histological cell type, a range of other potentially prognostic parameters have been discovered in the past few years. Many of these have only been described once so that they cannot be considered established markers. A few, however, such as vascular patterns or monosomy 3, were independently identified by several groups and now constitute recognized prognostic markers. The association of these factors with the disease course provides us with ever-new insights into the biology of this tumor. In particular, with the aid of new technologies such as microarray analysis, researchers around the globe hope that new and exciting discoveries will be made that can also modify therapy concepts.

Comprehensive methylation analysis in typical and atypical PWS and AS patients with normal biparental chromosomes 15.

Imprinting defects in 15q11-q13 are a rare but significant cause of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Patients with an imprinting defect have apparently normal chromosomes 15 of biparental origin, but are recognised by @parental DNA methylation at D15S63 (PW71) or SNURF-SNRPN exon 1. We have investigated the methylation status of five additional loci in 12 such patients with or without a deletion in the imprinting centre. In each patient, the imprinting defect affected all loci tested. During routine diagnostic testing we identified four patients who had a normal methylation pattern at SNURF-SNRPN exon 1, but an abnormal pattern at D15S63. In two of these patients, who were suspected of having PWS, this change was restricted to D15S63. In two patients suspected of having AS, several but not all loci were affected. Using a newly developed methylation-specific PCR test for D15S63 we found that these methylation changes are rare in patients suspected of having AS. Although we can not prove that the methylation changes in the four patients are causally related to their disease, our findings demonstrate that spatially restricted changes in methylation can occur. In some cases, these changes may reflect incomplete imprint spreading.

Partial deletions of the long and short arm of chromosome 3 point to two tumor suppressor genes in uveal melanoma.

Uveal melanoma is the most common form of primary eye cancer. Monosomy 3, which is an unusual finding in tumors but is present in approximately 50% of uveal melanomas, is significantly correlated with metastatic disease. To obtain positional information on putative tumor suppressor genes on this chromosome, we have investigated tumors from 333 patients by comparative genomic hybridization, microsatellite analysis, or conventional karyotype analysis. A partial deletion of the long arm was found in eight tumors, and the smallest region of deletion overlap (SRO) spans 3q24-q26. We found six tumors with a partial deletion of the short arm and were able to define a second SRO of about 2.5 Mb in 3p25. This SRO does not overlap with the VHL gene. Our finding suggests a role for two tumor suppressor genes in metastasizing uveal melanoma and may explain the loss of an entire chromosome 3 in these tumors.

Identification of chromosomes 3, 6, and 8 aberrations in uveal melanoma by microsatellite analysis in comparison to comparative genomic hybridization.

In uveal melanoma, monosomy 3 is strongly associated with metastic disease and poor prognosis. Cytogenetic analysis and comparative genomic hybridization (CGH) have been used to identify chromosomal aberrations in uveal melanoma. As these methods are costly and time consuming in routine diagnostic settings, we evaluated whether tumors with monosomy 3 can be reliably identified by microsatellite analysis (MSA). In addition, we also tested if aberrations of chromosomes 6 and 8, which have also been associated with the course of the disease, can be detected by MSA. We established a protocol for MSA of 23 markers, 3-4 on each arm of chromosomes 3, 6, and 8. Twenty tumors were analyzed by CGH and MSA, and 10 tumors were analyzed by MSA only. For chromosome 3, the results of CGH and MSA were concordant, thus indicating that the dosage of this chromosome can reliably be determined by MSA. However, MSA failed to detect copy number gains at 6p in some tumors. Moreover, despite quantitative evaluation of allele ratios, it was not possible to discern 8p losses and gains reliably. We thus conclude that while MSA can be used to determine monosomy 3 in uveal melanoma, careful interpretation of results for chromosomes 6 and 8 is recommended.

Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer.

BACKGROUND: A growing body of evidence supports the hypotheses that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene and that the chemopreventive effects of retinoids are due to induction of RAR-beta2. RAR-beta2 expression is reduced in many malignant tumors, and we examined whether methylation of RAR-beta2 could be responsible for this silencing. METHODS: RAR-beta2 expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in eight breast cancer cell lines that were either treated with the demethylating agent 5-aza-2'-deoxycytidine and subsequently with all-trans-retinoic acid (ATRA) or left untreated. Sodium bisulfite genomic sequencing was used to determine the locations of 5-methylcytosines in the RAR-beta2 genes of three of these cell lines. In 16 breast cancer biopsy specimens and non-neoplastic breast tissue, methylation-specific PCR was used to determine the methylation status of RAR-beta2, and, in 13 of the specimens, RT-PCR analysis was used to detect RAR-beta2 expression. RESULTS: Cell lines SK-BR-3, T-47D, ZR-75-1, and MCF7 exhibited expression of RAR-beta2 only after demethylation and treatment with ATRA. The first exon expressed in the RAR-beta2 transcript was methylated in cell lines ZR-75-1 and SK-BR-3. Six breast cancer specimens showed methylation in the same region of the gene. No expression of RAR-beta2 was found in any grade III lesion. An inverse association between methylation and gene expression was found in all grade II lesions. The RAR-beta2 gene from non-neoplastic breast tissue was unmethylated and expressed. CONCLUSIONS: Methylation of the RAR-beta2 gene may be an initial step in breast carcinogenesis; treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality.

Methylation mosaicism of 5'-(CGG)(n)-3' repeats in fragile X, premutation and normal individuals.

Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5'-(CGG)(n)-3' repeat in the promoter and 5'-untranslated region (5'-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M- Sss I-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5'-(CGG)(n)-3' repeats and in the levels of methylation in the repeat and the 5'-UTR. In one patient (OEl) with high repeat length hetero-geneity ( n = 15 to >200), shorter repeats (n = 20-80) were methylated or unmethylated, longer repeats ( n = 100-150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5'-CG-3' sequences were found in some repeats and 5'-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M- Sss I-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.

Detection of homozygous deletions in tumors by hybridization of representational difference analysis (RDA) products to chromosome-specific YAC clone arrays.

Representational difference analysis (RDA), a subtractive hybridization method that enriches differences between complex genomes, can be used to isolate fragments deleted in tumor genomes. Usually, most of the clones obtained by this approach result from polymorphic fragments. Therefore, identification of homozygously deleted fragments, which can indicate the presence of tumor suppressor loci, is often tedious. To overcome this limitation, we devised a novel strategy in which labeled RDA products are hybridized in toto against membranes spotted with YAC clones covering a region of interest. In such a way, identified YAC clones provide positional information on homozygous deletions and loss of heterozygosity (LOH) regions. We have tested this approach with a tumor known to have a homozygous deletion within a region of LOH on chromosome 13. RDA was performed using representations generated with restriction enzymes Bgl II, Nco I and Xba I, and the difference products of each experiment were separately hybridized to chromosome 13 YAC filters. When collating the map positions of positive YACs from three different RDA experiments a cluster of hits clearly identified the region on chromosome 13 which comprised the homozygous deletion. This shows that our novel approach can be effective.

A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus.

The analysis of allelic methylation differences in 15q11-q13 has been established as a valid test for the Angelman and Prader-Willi syndromes. Current tests use methylation-sensitive restriction enzymes and Southern blot analysis. Here we describe a single-tube PCR test. It is based on sodium bisulfite treatment of DNA, which converts unmethylated, but not methylated cytosine residues to uracil, and PCR primers specific for the maternal and the paternal allele. The method was validated in a blinded retrospective study on 87 DNA samples from normal controls and patients. Prospective studies by independent laboratories will be needed before this assay can replace Southern blot analysis in routine diagnostic procedures.

Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method.

A deletion of 15q11-q13 and uniparental disomy 15 lead to Prader-Labhart-Willi syndrome (PWS) or Angelman syndrome (AS) because this region contains genes expressed exclusively from the paternal (PWS) or maternal (AS) chromosome, respectively. DNA methylation plays a role in the control of imprinted gene expression, but so far only a few 5'-CG-3' dinucleotides within the recognition sites of the methylation sensitive enzymes have been studied. As part of a study on DNA methylation patterns in the human genome, we have applied the bisulfite protocol of genomic sequencing to study all 5'-CG-3' dinucleotides around exon 1 of SNRPN and at the D15S63 locus, which contains a start site for alternative SNRPN transcripts possibly involved in imprint switching during gametogenesis. At least 17 PCR products derived from single chromosomes of normal individuals as well as PWS and AS patients have been sequenced. We have found that cytosine residues outside 5'-CG-3' dinucleotides are always unmethylated. However, > 96% of all of the 23 5'-CG-3' dinucleotides around SNRPN exon 1 are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome. This finding is in contrast to the D15S63 locus, where only the two Cfol/Hhal sites are methylated on the maternal chromosome at the same frequency as seen for the SNRPN segment. At the other five 5'-CG-3' dinucleotides, differential methylation is less pronounced, i.e. 45-70% on the maternal chromosome and 5-14% on the paternal chromosome. The differences between SNRPN and D15S63 methylation may reflect different biological functions of the alternative SNRPN transcripts. The systematic investigation of 5'-CG-3' methylation patterns as reported here will provide the basis for a PCR-based methylation test to diagnose PWS and AS.