Disruption of MBD5 contributes to a spectrum of psychopathology and neurodevelopmental abnormalities.

Microdeletions of chromosomal region 2q23.1 that disrupt MBD5 (methyl-CpG-binding domain protein 5) contribute to a spectrum of neurodevelopmental phenotypes; however, the impact of this locus on human psychopathology has not been fully explored. To characterize the structural variation landscape of MBD5 disruptions and the associated human psychopathology, 22 individuals with genomic disruption of MBD5 (translocation, point mutation and deletion) were identified through whole-genome sequencing or cytogenomic microarray at 11 molecular diagnostic centers. The genomic impact ranged from a single base pair to 5.4 Mb. Parents were available for 11 cases, all of which confirmed that the rearrangement arose de novo. Phenotypes were largely indistinguishable between patients with full-segment 2q23.1 deletions and those with intragenic MBD5 rearrangements, including alterations confined entirely to the 5'-untranslated region, confirming the critical impact of non-coding sequence at this locus. We identified heterogeneous, multisystem pathogenic effects of MBD5 disruption and characterized the associated spectrum of psychopathology, including the novel finding of anxiety and bipolar disorder in multiple patients. Importantly, one of the unique features of the oldest known patient was behavioral regression. Analyses also revealed phenotypes that distinguish MBD5 disruptions from seven well-established syndromes with significant diagnostic overlap. This study demonstrates that haploinsufficiency of MBD5 causes diverse phenotypes, yields insight into the spectrum of resulting neurodevelopmental and behavioral psychopathology and provides clinical context for interpretation of MBD5 structural variations. Empirical evidence also indicates that disruption of non-coding MBD5 regulatory regions is sufficient for clinical manifestation, highlighting the limitations of exon-focused assessments. These results suggest an ongoing perturbation of neurological function throughout the lifespan, including risks for neurobehavioral regression.

Non-linear registration improves statistical power to detect hippocampal atrophy in aging and dementia.

OBJECTIVE: To compare the performance of different methods for determining hippocampal atrophy rates using longitudinal MRI scans in aging and Alzheimer's disease (AD). BACKGROUND: Quantifying hippocampal atrophy caused by neurodegenerative diseases is important to follow the course of the disease. In dementia, the efficacy of new therapies can be partially assessed by measuring their effect on hippocampal atrophy. In radiotherapy, the quantification of radiation-induced hippocampal volume loss is of interest to quantify radiation damage. We evaluated plausibility, reproducibility and sensitivity of eight commonly used methods to determine hippocampal atrophy rates using test-retest scans. MATERIALS AND METHODS: Manual, FSL-FIRST, FreeSurfer, multi-atlas segmentation (MALF) and non-linear registration methods (Elastix, NiftyReg, ANTs and MIRTK) were used to determine hippocampal atrophy rates on longitudinal T1-weighted MRI from the ADNI database. Appropriate parameters for the non-linear registration methods were determined using a small training dataset (N = 16) in which two-year hippocampal atrophy was measured using test-retest scans of 8 subjects with low and 8 subjects with high atrophy rates. On a larger dataset of 20 controls, 40 mild cognitive impairment (MCI) and 20  AD patients, one-year hippocampal atrophy rates were measured. A repeated measures ANOVA analysis was performed to determine differences between controls, MCI and AD patients. For each method we calculated effect sizes and the required sample sizes to detect one-year volume change between controls and MCI (NCTRL_MCI) and between controls and AD (NCTRL_AD). Finally, reproducibility of hippocampal atrophy rates was assessed using within-session rescans and expressed as an average distance measure DAve, which expresses the difference in atrophy rate, averaged over all subjects. The same DAve was used to determine the agreement between different methods. RESULTS: Except for MALF, all methods detected a significant group difference between CTRL and AD, but none could find a significant difference between the CTRL and MCI. FreeSurfer and MIRTK required the lowest sample sizes (FreeSurfer: NCTRL_MCI = 115, NCTRL_AD = 17 with DAve = 3.26%; MIRTK: NCTRL_MCI = 97, NCTRL_AD = 11 with DAve = 3.76%), while ANTs was most reproducible (NCTRL_MCI = 162, NCTRL_AD = 37 with DAve = 1.06%), followed by Elastix (NCTRL_MCI = 226, NCTRL_AD = 15 with DAve = 1.78%) and NiftyReg (NCTRL_MCI = 193, NCTRL_AD = 14 with DAve = 2.11%). Manually measured hippocampal atrophy rates required largest sample sizes to detect volume change and were poorly reproduced (NCTRL_MCI = 452, NCTRL_AD = 87 with DAve = 12.39%). Atrophy rates of non-linear registration methods also agreed best with each other. DISCUSSION AND CONCLUSION: Non-linear registration methods were most consistent in determining hippocampal atrophy and because of their better reproducibility, methods, such as ANTs, Elastix and NiftyReg, are preferred for determining hippocampal atrophy rates on longitudinal MRI. Since performances of non-linear registration methods are well comparable, the preferred method would mostly depend on computational efficiency.

Inter-observer variation of hippocampus delineation in hippocampal avoidance prophylactic cranial irradiation.

BACKGROUND: Hippocampal avoidance prophylactic cranial irradiation (HA-PCI) techniques have been developed to reduce radiation damage to the hippocampus. An inter-observer hippocampus delineation analysis was performed and the influence of the delineation variability on dose to the hippocampus was studied. MATERIALS AND METHODS: For five patients, seven observers delineated both hippocampi on brain MRI. The intra-class correlation (ICC) with absolute agreement and the generalized conformity index (CIgen) were computed. Median surfaces over all observers' delineations were created for each patient and regional outlining differences were analysed. HA-PCI dose plans were made from the median surfaces and we investigated whether dose constraints in the hippocampus could be met for all delineations. RESULTS: The ICC for the left and right hippocampus was 0.56 and 0.69, respectively, while the CIgen ranged from 0.55 to 0.70. The posterior and anterior-medial hippocampal regions had most variation with SDs ranging from approximately 1 to 2.5 mm. The mean dose (Dmean) constraint was met for all delineations, but for the dose received by 1% of the hippocampal volume (D1%) violations were observed. CONCLUSION: The relatively low ICC and CIgen indicate that delineation variability among observers for both left and right hippocampus was large. The posterior and anterior-medial border have the largest delineation inaccuracy. The hippocampus Dmean constraint was not violated.

Expression of the stem cell self-renewal gene Hiwi and risk of tumour-related death in patients with soft-tissue sarcoma.

Self-renewal is considered as a common property of stem cells. Dysregulation of stem cell self-renewal is likely a requirement for the development of cancer. Hiwi, the human Piwi gene, encodes a protein responsible for stem cell self-renewal. In this study, we investigated the expression of Hiwi at the RNA level by real-time quantitative PCR in 65 primary soft-tissue sarcomas (STS) and ascertained its impact on prognosis for STS patients. In a multivariate Cox's proportional hazards regression model, we found that an increased expression of Hiwi mRNA is a significant negative prognostic factor for patients with STS (P=0.017; relative risk 4.6, 95% confidence interval (CI) 1.3-16.1) compared to medium expression of Hiwi transcript. However, a low expression of Hiwi transcript is correlated with a 2.4-fold (CI 0.7-8.0) increased risk, but this effect was not significant (P=0.17). Altogether, high-level expression of Hiwi mRNA identifies STS patients at high risk of tumour-related death. This is the first report showing a correlation between expression of a gene involved in stem cell self-renewal and prognosis of cancer patients.

Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients.

Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyse stem cell-associated gene expression. We investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time-PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. Coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.

Mouse models in the study of the Ets family of transcription factors.

The Ets family of transcription factors is one of a growing number of master regulators of development. This family was originally defined by the presence of a conserved DNA binding domain, the Ets domain. To date, nearly 30 members of this family have been identified and implicated in a wide range of physiological and pathological processes. Despite the likely importance of Ets-family members, each of their precise roles has not been delineated. Herein, we describe the elucidation of essential functions of a few of these family members in vivo using knockout mouse models. Of the knockouts generated to date, the majority shows important functions in hematopoiesis, ranging from PU.1, a principle regulator of myelo-lymphopoiesis, to Spi-B which regulates the proper function of terminally differentiated cells. Ets1 was shown to be of intermediate importance as a regulator of pan-lymphoid development. Other Ets family members such as Fli1 and TEL1 display distinct and/or overlapping functions in vasculo/angiogenesis, hemostasis and hematopoiesis. The remaining knockouts generated, Ets2 and Er81, show non-hematopoietic defects related to extraembryonic development and neurogenesis, respectively. The pioneering group of knockout models described reveals only the most distinct functions of each of these Ets family members. A better understanding of the roles and hierarchies of Ets family members in cellular differentiation will come with the generation of new null alleles in previously untargeted family members, more mutant alleles in members already disrupted, double knockouts, ES cell differentiation and chimera rescue experiments, and tissue-specific inducible knockouts.

TRIM25 has a dual function in the p53/Mdm2 circuit.

P53 is an important tumor suppressor that, upon activation, induces growth arrest and cell death. Control of p53 is thus of prime importance for proliferating cells, but also for cancer therapy, where p53 activity contributes to the eradication of tumors. Mdm2 functionally inhibits p53 and targets the tumor suppressor protein for degradation. In a genetic screen, we identified TRIM25 as a novel regulator of p53 and Mdm2. TRIM25 increased p53 and Mdm2 abundance by inhibiting their ubiquitination and degradation in 26 S proteasomes. TRIM25 co-precipitated with p53 and Mdm2 and interfered with the association of p300 and Mdm2, a critical step for p53 polyubiquitination. Despite the increase in p53 levels, p53 activity was inhibited in the presence of TRIM25. Downregulation of TRIM25 resulted in an increased acetylation of p53 and p53-dependent cell death in HCT116 cells. Upon genotoxic insults, TRIM25 dampened the p53-dependent DNA damage response. The downregulation of TRIM25 furthermore resulted in massive apoptosis during early embryogenesis of medaka, which was rescued by the concomitant downregulation of p53, demonstrating the functional relevance of the regulation of p53 by TRIM25 in an organismal context.

Low detection rate of p53 antibodies in sera of soft tissue sarcoma patients.

Accumulation of the p53 gene product can lead to its immunogenic appearance and the generation of p53 serum antibodies (p53ab). In different cancer types the occurrence of detectable p53ab has an independent prognostic impact. In spite of the known p53 protein overexpression rate in soft tissue sarcomas (STS), up to 70%, there have been no investigations done on p53ab in serum in STS patients. In this prospective study of 50 STS patients, we investigated the presence of serum p53ab using an enzyme-linked immunosorbent assay system and the presence of p53 overexpression in the appropriate tissue specimen immunohistochemically. Using Kruskal-Wallis chi(2) and Kaplan-Meier tests the results were then correlated to histopathological and clinical data. Six of the 50 patients (12%) showed p53ab detectable in the serum, and 56% (28/50) of the tumors were p53 immunohistochemically positive. Four of the six p53ab positives (66%) had immunohistochemically p53 positive and two (33%) had negative tumors. Altogether four of the 50 patients (8%) were positive for p53ab in serum as well as for p53 immunohistochemistry in tumor tissue specimens. Twenty patients (40%) were negative for both. All of the p53ab positive patients had stage I or II tumors. Excluding tumor stage there was no p53ab correlation to histopathological, clinical or prognostic parameters. We conclude that in STS patients, p53ab also occurs but in contrast to other tumor types at a relatively low frequency. According to our results, the clinical value of p53ab seems to be limited in STS patients.

Gains in chromosomes 7, 8q, 15q and 17q are characteristic changes in malignant but not in benign peripheral nerve sheath tumors from patients with Recklinghausen's disease.

In order to investigate typical genomic alterations in patients with Recklinghausen's disease (NF1) we studied one from each of the six patients with NF1 several benign and/or malignant tumors. By means of comparative genomic hybridization (CGH) gained results from six benign neurofibromas and 14 malignant peripheral nerve sheath tumors (MPNSTs) were compared with four benign peripheral nerve sheath tumors (BPNSTs) from patients without NF1. In all 14 MPNSTs DNA sequence copy number changes were detected with a mean value of 13.5 imbalances per sample. The most frequent gains were in 8q, 17q (12 tumors each), 7p, 15q (ten tumors each), and 7q (nine tumors). We found ten high-level amplifications in nine of the 14 samples. In two cases, the high-level amplification involved 7p14-pter and 17q24-qter as well. The most frequent loss was in 17p (seven tumors). The benign neurofibromas from NF1-patients and the sporadic BPNSTs revealed only partially DNA sequence copy number changes without any distinct pattern. The gains of #7, 8q, 15q, and 17q were found exclusively in MPNSTs but not in neurofibromas and are supposed to be associated with malignant tumor progression. In comparison of the results of the 14 MPNSTs from NF1-patients with the results of previously published 20 sporadic MPNSTs, we found that the gain of 8q occurs most frequently in both tumor groups. Of course additionally in the sporadic MPNSTs there were more frequent gains of 5p, #6, and statistically significant gains of 20q. On the other hand in the MPNSTs from NF1-patients the most frequent gains were found in #7, and statistically significant in 15q, and 17q.

Colony formation of soft tissue sarcoma cells is inhibited by lipid-mediated antisense oligodeoxynucleotides targeting the human mdm2 oncogene.

More than one third of human soft tissue sarcoma (STS) have elevated levels of the MDM2 oncoprotein, resulting either from gene amplification or alternate mechanisms. MDM2 functions as a negative feedback regulator of the tumor suppressor p53. The aim of the present study was to investigate whether mdm2-antisense oligodeoxyribonucleotides (AS-ODNs) can influence the growth characteristics of two MDM2-overexpressing STS cell lines (US8-93, LMS6-93) where both have heterozygous p53 non-missense mutations. Cells were treated with lipofectamine-complexed mdm2 AS-ODNs complementary to a sequence of the mdm2 cDNA initiation site in comparison to sense control ODNs. After seeding and cultivation of a defined cell number the clonogenic survival was performed. The treatment of US8-93 cells with AS-ODNs, but not with sense ODNs, decreased the number of colonies up to > 80%. Western blot analysis demonstrated a significant decreasing of MDM2 protein level in AS-ODN transfected cells indicating an AS-specific inhibition of mdm2 transcription in US8-93 cells. Additionally, an increase of the G2/M population was found. In contrast, in the LMS6-93 cells treated with AS-ODNs only a decrease in clonogenic survival up to 26%, no change in MDM2 protein level and no cell cycle alterations were seen. All these factors taken together into consideration can be suggest that lipid-mediated mdm2 AS-ODNs could be as an effective therapeutic strategy for STS with an abnormal mdm2 overexpression.

Expression of alternatively and aberrantly spliced transcripts of the MDM2 mRNA is not tumor-specific.

The MDM2 proto-oncogene encodes a 90-kDa protein that binds to and inactivates the tumor suppressor p53. Several reports describe the presence of different alternatively, as well as, aberrantly spliced transcripts of the MDM2 mRNA in a variety of human cancers that have lost the ability to bind p53. Due to the transforming ability of at least some of the isoforms it has been suggested that they might contribute to tumorigenesis. Here we show that shorter MDM2 transcripts are also widely expressed in normal tissues, including lung and renal tissue, and in lymphocytes. Alteration in MDM2 RNA transcripts were found in the majority of the samples. Although we cannot exclude that alterations in MDM2 preferentially occur during cancer development, our data rather indicate that in this context the commonly observed transcript variants may also possess a normal physiological function.

Association of p53 mutations, microvessel density and neoangiogenesis in pairs of colorectal cancers and corresponding liver metastases.

p53 suppressor gene mutations are a well known step which occurs in the late stages of the complex tumourigenesis of colorectal cancer. A deregulation of p53 protein function may be associated with increased neovascularization and aggressive tumour growth. In vitro studies have shown that these genetic alterations cause a loss of wild-type p53-induced anti-angiogenetic control and could possibly induce expression of the neoangiogenic vascular endothelial growth factor (VEGF). Therefore, this in vivo study was performed to assess p53 mutations, i.e. hot spots in exons 4-9, in primary colorectal cancers and in corresponding liver metastases in order to test whether there is an association between p53 mutated tumours with increased microvessel density (MVD) and VEGF overexpression. Twenty-two tissue samples taken from primary colorectal cancers and the corresponding liver metastases were immediately snap-frozen in liquid nitrogen and fixed in formaldehyde. After DNA extraction exons 4-9 were amplified and directly sequenced. Cryostat sections were stained immunohistochemically using antibodies against VEGF, CD34, and p53 protein. A modified semiquantitative Weidner score and interactive computerized image analysis was used to assess MVD. Overexpression of immunohistochemically detected p53 protein was found in 7 of the 11 primary tumours and liver metastases (64%). Sequencing showed 3 out of 11 primary tumours (27%) and 5 out of 11 liver metastases (46%) to have p53 point or frameshift mutations; these samples tested immunohistochemically positive for p53 protein. Two p53 mutations in samples of liver metastases were not detectable in the corresponding primaries. We detected one frameshift mutation in exon 4 that has not yet been described in the literature. Tumour samples with p53 mutations and increased VEGF immunoreactivity were associated with higher MVD (p<0.01 and p<0.05, respectively). However, there was no association detected immunohistochemically between p53 and MVD as well as p53 mutations and VEGF overexpression. Our data demonstrate specific genetic alterations in the coding regions of p53 suppressor gene in both primary colorectal cancers and corresponding liver metastases, these alterations are associated with an increase in MVD, but not in VEGF overexpression. In addition, a novel frameshift mutation in both colorectal cancer and metastasis is described.

Expression of cathepsin B, D and L protein in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is the most common childhood autoimmune rheumatic disease and like rheumatoid arthritis (RA), it is characterized by inflammation and the progressive destruction of joints. In RA, cathepsins as proteinases play a major role in destroying synovial tissue and cartilage matrix. So far no data on cathepsin expression in pannus tissue of HA patients exist. The aim of this study was to characterize the expression levels of cathepsins B, D, H, and L in HA and to compare them with those in RA. Synovectomy tissue from 16 HA and 12 RA patients was investigated for cathepsin expression levels by Western blot analysis. Expression of cathepsins B, D and L was on comparable levels in the synovectomy tissue of HA and RA patients. The following graduation of expression was determined: cathepsin D > cathepsin L > cathepsin B. Cathepsin H was neither found to be expressed in HA nor in RA patients. The expression levels of cathepsins in pannus tissue showed no clear difference between patients with systemic JIA and patients with monoarticular JIA. In summary, the comparable expression of cathepsins B, D and L in RA and JIA synovectomy tissue suggests that they may play a similarly important role in destroying synovial tissue and cartilage matrix in the course of HA and RA.

Cytogenetic characterization of six malignant peripheral nerve sheath tumors: comparison of karyotyping and comparative genomic hybridization.

We analysed six malignant peripheral nerve sheath tumors (MPNSTs) from four patients using metaphase preparations and compared the results with those obtained by using comparative genomic hybridization (CGH). All six tumors showed structural and numerical chromosomal aberrations, mostly of chromosomes 1, 5, 7-10, 14-17, 19, 21, and 22. The number of chromosomes per tumor cell ranged from 42 to 104. We could not find a recurrent specific pattern of structural changes after comparing the MPNSTs of different patients. However, aberrations of different tumors from the same patient were nearly identical. In the four patients, we found a total of 117 breakpoints, mostly in 21q11.2 (seven times), in 8q11.2 and 14q10 (six times each), in 5q11.2 and 15q26 (four times each), in 8p11.2, 10q11.2, 16q22, 19q13.3, and 22q10 (three times each). In three MPNSTs, double minute chromosomes (dmin) we detected with metaphase investigations and high-level amplifications by using CGH, respectively. C-MYC gene amplification and loss of the P53 gene could be ruled out by locus-specific probes for the common gain of 8q and for losses of 17p. When comparing the CGH results with those of karyotyping an overlap in the most frequent gains in 7q, 8q, 15q, and 17q was observed. However, we found more frequent losses in 19q in the metaphase investigations.

Novel mdm2 splice variants identified in pediatric rhabdomyosarcoma tumors and cell lines.

Mdm2 is an oncogene that binds to and inactivates the tumor suppressor p53. However, the presence of oncogenic splice variants of mdm2 in human tumors that lack the p53 binding site has suggested a p53-independent transforming function for this protein. This report describes expression of 11 different mdm2 splice variants in pediatric rhabdomyosarcoma (RMS) cell lines and tumors at a frequency of 75% and 82%, respectively. Five of these isoforms have previously been described in other tumor histiotypes but six are novel and may be unique to RMS. There was no association between expression of splice variants and mdm2 gene amplification or p53 status. In addition, the frequency of splice variants was much higher than the incidence of mdm2 amplification or p53 mutations. These variants may be important to consider with respect to RMS tumor progression and therapeutic response.

Radiosensitization in sarcoma cell lines with a p53 missense mutation correlates with prevention of irradiation G2/M arrest but not with induction of apoptosis.

We analysed the effects of caffeine and taxol on the radiobiological behaviour of two human sarcoma cell lines (RD, SK-LMS-1) each with a p53 missense mutation. Treatment with 2 mM caffeine resulted in an inhibition of the irradiation induced G2/M arrest in both cell lines. This effect was coupled with a radiosensitization in cell line SK-LMS-1 after an irradiation with 6 Gy (enhancement factor of 5.0). However, the effect of radiosensitization was not correlated with an induction of apoptosis. Incubation with 20 nM taxol increased the irradiation induced apoptosis almost 3-fold in cell line SK-LMS-1, but not in cell line RD. However, taxol had no effect on the irradiation induced G2/M arrest or radiosensitivity in either cell line. The results support the hypothesis that the prevention of irradiation induced G2/M arrest but not the induction of apoptosis plays a critical role in determining radiosensitivity in sarcoma cell lines with p53 mutations.

Transfection with mdm2-antisense or wtp53 results in radiosensitization and an increased apoptosis of a soft tissue sarcoma cell line.

Soft tissue sarcomas (STS) are mostly resistant after radiation treatment and are characterized by a rather low rate of apoptosis. The aim of this study was to test, in the p53 mutant STS cell line US8-93, the effect of a combined treatment with DNA transfection--either with mdm2 antisense oligodesoxynucleotides (mdm2-AS) or with a wild-type p53-plasmid (wtp53)--and the effects of irradiation on radiosensitivity. Mdm2-sense oligodesoxynucleotides (mdm2-SE) and a GFP-plasmid (GFP) were applied as controls. In order to evaluate the treatment radiation sensitization (clonogenic survival), apoptotic cell death and P53/MDM2-protein expression were determined. A moderately increased radiation sensitization was observed when comparing clonogenic survival after 2 Gy irradiation between cells transfected either with the control mdm2-SE (48%) or with mdm-2 AS (30%). At the same irradiation dose, clonogenic survival of wtp53-plasmid transfected cells (32%) was about 2-fold less than in the cells transfected with the control GFP-plasmid (61%). This enhancement factor of radiation sensitization was increased by about 3-fold at 4 Gy irradiation. Furthermore, an increase in apoptotic cells was already detectable by up to 7.7% (mdm2-AS) in comparison to 3.1% (mdm2-SE control) 72 hours after transfection. In parallel, the percentage of apoptotic cells could be further elevated after subsequent irradiation with 12 Gy by up to 15% (mdm2-AS) compared to 5.7% (mdm2-SE control). A striking result was obtained with the combined treatment of a wtp53 and 12 Gy irradiation which produced in 25% and 38.9% of apoptotic cells 48 hours and 72 hours after transfection, respectively. We can therefore conclude that the sensitivity of radiation therapy is enhanced by DNA transfection with wtp53 or mdm-2 AS ODNs for the correction of the p53-mdm2 balance in STS in vitro.

A MboII polymorphism in exon 11 of the human MDM2 gene occuring in normal blood donors and in soft tissue sarcoma patients: an indication for an increased cancer susceptibility?

The human MDM2 oncogene, well known as the tumor suppressor gene p53's partner, plays an important role in tumorigenesis whether it is dependent on or independent of TP53. In this study, we investigated in a PCR-sequencing analysis the exon 11 of the human MDM2 gene for gene alterations. A MboII polymorphism occurs in 8% of normal blood donors (8 out of 100 probands) and in 13% of the soft tissue sarcoma patients (11 out of 82 patients). Of note was that two STS patients carried the gene alteration only in the tumor specimens heterozygously but not in normal tissue. In a Kaplan-Meier analysis, patients without the polymorphism, indicated a median survival rate of 57 months, whereas, patients with the polymorphism survived on average only 38 months. We suggest that this polymorphism might be associated with an increased cancer susceptibility.

[Both somatic and germline genetics of the TP53-pathway influence ovarian cancer incidence and survival]. / Einfluss von Mutationen und Polymorphismen im TP53-Tumorsuppressorpathway auf die Entstehung und Prognose von Ovarialkarzinomen.

PURPOSE: Although TP53 is one of the most studied genes/proteins in ovarian carcinomas, the predictive value of TP53 alterations is still ambiguous. EXPERIMENTAL DESIGN: We performed analyses of the TP53 mutational status and its protein expression by immunohistochemistry. Moreover, the single nucleotide polymorphism SNP309 in the P2-promotor of the HDM2 gene was investigated. We correlated the results with the age of onset and the outcome of 107 ovarian carcinoma patients. RESULTS: In our study, we identified a large group of patients with TP53 overexpression despite having a wild-type gene (49% of all patients with wild-type TP53). This was associated with a significantly shortened overall survival time (p = 0.019). Patients with TP53 alterations (especially those with overexpression of wild-type TP53) were also more refractory to chemotherapy than patients with normal TP53 (p = 0.027). The Gallele of the SNP309 is associated with an earlier age of onset in estrogen receptor expressing FIGO stage III patients (p = 0.048). In contrast, in FIGO III patients, a weakened TP53 pathway (either G-allele of SNP309 or a TP53 mutation) is correlated with an increased overall survival compared with patients whose tumors are wild-type for TP53 and SNP309 (p = 0.0035). CONCLUSION: Our study provides evidence that both germ line and somatic alterations of the TP53 pathway influence incidence and survival of ovarian carcinoma, and it underscores the importance of assessing the functionality of TP53 in order to predict sensitivity of platin-based chemotherapies and patient outcome.

The effects of knockdown of wild-type survivin, survivin-2B or survivin-delta3 on the radiosensitization in a soft tissue sarcoma cells in vitro under different oxygen conditions.

The inhibitor of apoptosis wild-type survivin is a multifunctional protein that suppresses apoptosis and regulates cell cycle progression. An association between wild-type survivin expression and radiosensitivity has been described in different tumor cells. The effects of siRNA-induced knockdown of wild-type survivin and survivin-splice variants survivin-2B and survivin-Delta3 were investigated under normoxic and hypoxic conditions in the human sarcoma cell line US 8-93 (mutant p53). Inhibition of the survivin isoforms by siRNA resulted in a decrease of target mRNA down to 14-70% compared to cells treated with control siRNA independent of the oxygen level. The mRNA expression of survivin isoforms was decreased by the factor of 1-12 when the cells were cultivated under hypoxic conditions. Moreover, the knockdown of wild-type survivin reduced colony formation independent of oxygen concentration down to 70% and induced formation of polyploid cells. Less reduction of plating efficiency was observed after specific knockdown of survivin-2B and survivin-Delta3 under hypoxic or normoxic conditions. A knockdown of wild-type survivin, survivin-Delta3 and survivin-2B isoforms in combination with irradiation caused no radiosensitization in cell line US 8-93, neither under hypoxic nor under normoxic conditions tested in the colony-forming assay. However, knockdown of wild-type survivin caused radiosensitization in the megacolony assay.