Christian Hartmann

Genetic Signature of Oligoastrocytomas Correlates with Tumor Location and Denotes Distinct Molecular Subsets

Oligoastrocytomas are heterogeneous tumors that have molecular features that overlap with either oligodendrogliomas or astrocytomas. Differences in the frequency of chromosomal losses of 1p and 19q in oligodendrogliomas are related to tumor location, with a low rate of allelic loss in tumors of the temporal and a high rate in tumors of the frontal, parietal, and occipital lobes. To test the possibility of regional molecular heterogeneity in oligoastrocytoma, we examined a series of 203 gliomas including 68 oligoastrocytomas and two control groups of 73 oligodendrogliomas and 62 astrocytomas for allelic losses of chromosomal arms 1p and 19q, and TP53 mutations, and compared these data with tumor localization. Common molecular alterations were found in oligodendrogliomas and oligoastrocytomas arising in extratemporal sites. In respect to the molecular parameters analyzed, temporal oligoastrocytomas were either indistinguishable from astrocytoma or similar to temporal oligodendrogliomas. Oligodendroglial neoplasms can thus be separated into three molecular subsets, two of which include lesions with the morphological features of oligodendrogliomas and oligoastrocytomas and one resembling temporal oligoastrocytoma. Molecular subclassification thus unifies previous findings about prognosis, behavior, response to therapy, genotype, and location in oligodendroglial tumors.

A Prospective Evaluation of a Diffractive versus a Refractive Designed Multifocal Intraocular Lens

OBJECTIVE To evaluate prospectively a diffractive (811E, Pharmacia; power add +4.0 D) versus a refractive (PA154N, Allergan; power add +3.5 D) designed multifocal lens. PARTICIPANTS Eighty patients planned for cataract surgery without additional ocular pathologies were randomized into the diffractive or refractive group, respectively. INTERVENTION A standardized no-stitch phacoemulsification with implantation of one of the two multifocal lenses was performed in each patient. MAIN OUTCOME MEASURES Distance and near-visual acuity, contrast sensitivity, low contrast visual acuity, glare visual acuity, and depth of focus were measured after surgery. RESULTS All treated patients had best-corrected visual acuities of 20/30 or better. Near-uncorrected vision was significantly better (P < 0.0001) with the diffractive lens (mean, J1) than with the refractive lens (mean, J4). Low contrast visual acuity (61 +/- 12% versus 59 +/- 9%), glare visual acuity (39 +/- 19% versus 38 +/- 14%), and contrast sensitivity (1.48 +/- 0.08 versus 1.50 +/- 0.12) were not significantly different between the groups. CONCLUSIONS Both lens designs showed satisfactory functional results with advantages for the diffractive lens design.

Multiple meningiomas: Investigating the molecular basis of sporadic and familial forms.

Meningiomas are common tumors of the coverings of the central nervous system (CNS), comprising 20% of intracranial neoplasms. The only genes known to be associated with sporadic meningiomas are NF2 on chromosome 22 and the related cytoskeleton element DAL‐1 on chromosome 18. Between 1 and 8% of patients with meningiomas develop multiple meningiomas, a trait transmitted occasionally in an autosomal dominant fashion. We investigated the DAL‐1 and NF2 loci in 7 unrelated multiple meningioma patients without clinical evidence of NF2 by mutational and pathological analysis. Five novel intragenic microsatellite polymorphisms were developed for specific detection of loss of heterozygosity (LOH) at the DAL‐1 locus. Three of 7 patients had affected relatives and all affected individuals were female. No tumors from familial patients were of a fibroblastic subtype. Truncating NF2 mutations were detected in 3 tumor specimens, but were not present in the corresponding blood samples. Two tumors showed LOH at the NF2 locus. All tumors showing mutations at the NF2 locus originated from patients without affected relatives and were of the fibroblastic subtype. Five non‐truncating alterations in the DAL‐1 gene were found, however, LOH of chromosome 18 markers was not seen in any tumor. In contrast to the NF2 results, all DAL‐1 alterations were found in paired blood specimens. Our findings provide further evidence that the molecular basis of sporadic and familial multiple meningiomas is fundamentally different and extend this dichotomy to pathologic subtypes. DAL‐1 does not function as a true tumor suppressor in these patients.

Recombinant human basic fibroblast growth factor (Rh-bFGF) in three different wound models in rabbits: Corneal wound healing effect and pharmacology

Prior to a clinical trial in humans, we studied the effect and pharmacological distribution of recombinant human basic fibroblast growth-factor (Rh-bFGF) in vivo. Healing experiments on de-epithelialized rabbits corneas (n = 24 animals) compared the efficacy of three bFGF doses to controls and revealed a significantly increased healing rate for both 200 ng and 500 ng per application Rh-bFGF treatment groups compared to the control groups. To assess possible side effects of Rh-bFGF (500 ng topically applied for up to 7 days, twice daily), ten rabbits were involved in a model of an anterior keratectomy wound (performed with Draegers roto-keratome to a depth of 0.15 mm). Light microscopy of thin sections of treated corneas showed an increased fibrogenesis in the anterior stroma with a more pronounced activation of keratocytes. No evidence for abnormal neovascularization or inflammation was observed when compared to control corneas. Ocular penetration and systemic distribution of topically applied labelled 125I FGF was assessed in three models (iodine vapour epithelial burn, anterior keratectomy and penetrating autokeratoplasty) in 24 rabbits. No intraocular penetration of bFGF occurred as shown by direct gamma counting. Macroautoradiography showed a selective labelling of epithelial basement membrane when denuded and intact, as previously described. Evidence for systemic absorption of breakdown products was confirmed by heparin-sepharose chromatography of blood and urine samples. Under these conditions, we suggest that topical Rh-bFGF promotes corneal wound healing without morphological adverse reaction or intraocular and systemic penetration.

Subclassification of nerve sheath tumors by gene expression profiling

Nerve sheath tumors are the most common tumors of Neurofibromatosis type 1 (NF1) patients. Dermal neurofibromas develop in nearly all NF1‐patients, whereas plexiform neurofibromas are only observed in one‐third of the patients. NF1‐patients have about a 10% lifetime risk for developing malignant pheripheral nerve sheath tumors (MPNST). The origin of these tumors is thought to be the Schwann cell lacking functional neurofibromin. However, additional genetic alterations are likely to modulate tumor biology and to contribute to individual nerve sheath tumor entities. To gain insight into the molecular events and to determine whether these tumors can be classified according to gene expression profiles, we performed expression analysis applying cDNA array technology. Nine dermal neurofibromas, 7 plexiform neurofibromas, ten MPNST and two MPNST cell cultures were examined. All tumors but 6 sporadic MPNST were obtained from NF1‐patients. We detected significant differences in gene expression patterns between neurofibromas and MPNST and between dermal neurofibromas and plexiform neurofibromas. Tumor class prediction agreed in all but one case with histological and clinical classification. NF1‐associated and sporadic MPNST could not be distinguished by their gene expression patterns. We present a panel of discriminating genes that may assist subclassification of nerve sheath tumors.

Survival of corneal allografts following adenovirus-mediated gene transfer of interleukin-4.

Abstract Background: Genetic manipulation of the donor cornea ex vivo prior to transplantation may allow modulation of the allogeneic immune response following penetrating keratopasty. In this study we investigated the effect of adenovirus-mediated gene transfer of the Th2 cytokine interleukin-4 (IL-4) to rat corneas in an experimental keratoplasty model. Methods: Ex vivo manipulation of Wistar-Furth rat corneas was performed using E1/E3- deleted adenoviral vectors transferring the gene for rat IL-4 (AdrIL-4) under control of the CMV promoter. Following transfection with AdrIL-4 (2×108 pfu) in DMEM/2% FCS for 3 h, donor corneas were transplanted in MHC class I/II-incompatible Lewis rats. Fifty-two Lewis rats were randomly assigned to receive either nontransfected grafts (n=32), AdrIL-4-transfected grafts (n=8), or syngeneic grafts (n=12). Results: The rejection rate of AdrIL-4-transfected grafts (85.7%) could not be reduced as compared to controls (62.9%). In addition, the mean survival time of AdrIL-4-transfected grafts (12.6±4.5 days) did not differ (P>0.05) from that for untreated transplants (14.1±3.8 days). Conclusions: Our results indicate that overexpression of IL-4 is not sufficient to reduce the rejection rate of corneal allografts in an ex-perimental keratoplasty model. Further investigations are necessary to identify the reasons for failure and establish more efficient modulatory approaches.

Differentially expressed genes in neurofibromatosis 1-associated neurofibromas and malignant peripheral nerve sheath tumors

Neurofibromas represent one of the hallmarks of neurofibromatosis 1 (NF1) patients. Tumor progression of neurofibromas to malignant peripheral nerve sheath tumors (MPNST) is a frequent and life threatening complication. To learn more about processes involved in malignant transformation, we evaluated differential gene expression in plexiform neurofibroma and MPNST from the same NF1 patient. Suppression subtractive hybridization (SSH) yielded 133 differentially expressed genes confirmed by reverse Northern blotting. Virtual Northern blots were employed to validate 23 genes. To independently verify differential expression, immunohistochemical analyses with antibodies to matrix metalloproteinase 13 (MMP13), platelet-derived growth factor receptor alpha (PDGFRA) and fibronectin (FN1) were performed on 9 dermal and 9 plexiform neurofibromas and 16 MPNST from 19 NF1 patients. All three proteins proved to be up-regulated in MPNST. MMP13 expression was observed in 44% of MPNST but was absent in neurofibromas. PDGFRA was expressed in all tumors, but the number of cells expressing it was below 30% in neurofibromas and over 50% in MPNST. Likewise, FN1 was expressed in all tumors, but less than 30% of the cells in neurofibromas and more than 70% of the cells in MPNST exhibited antibody binding. Our data point to several genes not previously recognized to be differentially expressed, and provide a framework for future studies on progression-associated gene expression in low- and high-grade nerve sheath tumors.

Tumour necrosis factor alpha and interleukin 6 gene expression in keratocytes from patients with rheumatoid corneal ulcerations.

Background/aims: Ultrastructural alterations in the stroma adjacent to corneal perforations have previously been reported in patients with longstanding rheumatoid arthritis. Since patients with rheumatoid arthritis often present upregulation of proinflammatory cytokines in serum and in synovial fluid, it was of interest to analyse the gene expression of these cytokines—for example, tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), in corneal samples from patients with corneal ulcerations and/or perforations associated with rheumatoid arthritis. Methods: Corneal samples from seven patients with corneal ulcerations and/or perforations associated with rheumatoid arthritis were collected in 4% paraformaldehyde in “RNAse-free” conditions. Paraffin sections were fixed on silan coated slides and further analysed by systematic non-radioactive in situ hybridisation, using specific gene probes for TNF-α and IL-6 labelled with digoxigenin (DIG). Detection of hybrids was carried out by using a commercially available DIG detection system. Results: Whereas an extended TNF-α gene expression could be clearly observed in the keratocytes surrounding the corneal ulcerations and/or perforations from five of the seven analysed patients, all seven patients presented clearly positive results for an extended IL-6 gene expression in the analysed tissue samples. Conclusions: Alterations in corneal cells surrounding ulcerations and/or perforations in patients with rheumatoid arthritis may occur with implication for inflammatory processes. Upregulation of the proinflammatory cytokines TNF-α and IL-6 may modify the production of metalloproteinases in the corresponding cells resulting in collagenolytic corneal damage.

Intraocular antibody production in intraocular inflammation

Abstract Background: The production of intraocular antibodies is considered a specific marker for active infectious uveitis. The aim of our study was to evaluate the diagnostic value of aqueous humor analysis in consecutive patients referred to a tertiary clinical center. Methods: We analyzed 91 paired aqueous humor/serum samples from 89 patients with intraocular inflammation. In 71 patients aqueous humor analysis was used as a positive or negative confirmation of the suspected cause, whereas in 18 patients the clinical diagnosis was completely uncertain. A modified micro-ELISA technique was used to detect intraocular IgG production against Toxoplasma gondii, varicella zoster virus, herpes simplex virus and cytomegalovirus. Statistical analysis was performed using the ”Cohen’s kappa” test.Results: Specific intra-ocular antibody production could be detected in 12 (66.7%) of 18 patients with uncertain diagnosis. Subsequently initiated therapy led to clinical improvement in 10 patients, whereas 2 patients remained unchanged. In 2 (2.8%) of 71 patients aqueous humor analysis led to revision of the initially suspected etiology and to a change of therapy. Statistical analysis showed a significant accordance of diagnosis and aqueous humor analysis (P<0.01). Conclusion: In patients with infectious uveitis, analysis of intraocular synthesis of specific antibodies is a valuable tool to establish the etiology rapidly and allows initiation of targeted antimicrobial treatment.

Fine mapping of chromosome 22q tumor suppressor gene candidate regions in astrocytomas

Astrocytomas and glioblastomas are the most frequent primary brain tumors in adults. Mutations and altered expression of multiple genes have been found to contribute to the genesis of these tumors. However, many factors in the genesis of astrocytic gliomas are not resolved yet. The frequent losses on several chromosomes indicate the role of still unidentified tumor suppressor genes. Loss of heterozygosity (LOH) on 22q has been described in up to 30% of astrocytic tumors and may be associated with progression to anaplasia. In a first step, information from the nearly finished physical sequence of chromosome 22 were used to map LOH data from 22q deletion studies on different tumor entities to identify potential tumor suppressor gene candidate regions. Next, a series of 153 astrocytic gliomas was examined with 11 polymorphic markers spanning these regions. Forty‐nine (32%) astrocytic gliomas exhibited LOH on 22q, 17 (35%) of which lost heterozygosity for all markers and 32 (65%) of which carried interstitial or partial deletions. Two regions were identified on the physical DNA sequence. The centromeric region spans 3 Mb and the telomeric region 2.7 Mb. The reduced size of these regions now allows direct analysis of all genes included. We already performed mutation analysis on 4 candidate genes from these regions (MYO18B, DJ1042K10.2, MKL1 and EP300), but did not find any mutations in astrocytic tumors.