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.

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.

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.

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.

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.

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.

[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.

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.

[HDMX amplification and high levels of HDMX-S splice variant are correlated with a poor prognosis in soft tissue sarcomas]. / Die amplifikation des HDMX-Gens und insbesondere die Uberexpression der HDMX-S Spleissvariante korrelieren mit einer schlechten Prognose für Weichteilsarkompatienten.

The aim of this study was to determine the amplification status of the HDMX gene and the expression of the HDMX mRNA particularly that of the HDMX-S splice variant in soft tissue sarcomas (STS). We show that the HDMX gene is amplified in 27% of STSs, which was associated with a worse prognosis (RR = 2.8, p = 0.03). We have also found that the transcript of the HDMX-S variant was predominant in a subset of (14%) of tumor samples, which was correlated with a significantly decreased overall survival time (15 vs. 53 months, p < 0.0001, log-Rank-test) and with a 9-fold-increased risk of tumor-related death (p < 0.0001). There was no correlation between the HMDX gene amplification and the HDMX-S splice variant overexpression. In summary, our data indicate that both the overexpression of the HDMX-S transcript, as well as, the HDMX gene amplification are important prognostic markers for STS.

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.

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.

Gains of 12q are the most frequent genomic imbalances in adult fibrosarcoma and are correlated with a poor outcome.

Comparative genomic hybridization was used to analyze 41 adult fibrosarcomas from 34 patients. Thirty-one patients showed in their tumors DNA sequence copy number changes (mean 11, range 3-25). The minimal common regions for the most frequent gains were narrowed down to 12q21 (18 cases); 12q14-q15 and 14q22 (16 cases each); 4q22, 7q31, and 14q23-q24 (15 cases each); and 4q21, 4q23-q24, 8q22, and 12q22 (14 cases each). Twenty-five high-level amplifications were observed in 12 samples. 12q21 and 18p were affected three times each; and 1p21, 4q31.3, 7p21, 12q14-q15, Xp22.1-p22.2, and Xq22-q23 two times each. Losses were less frequent than gains. Early stages of adult fibrosarcomas were characterized by frequent gains of chromosomes 2, 4q, and 14q, whereas gains of chromosomes 7 and 8q were associated with progression. Gains of 12q were frequent in all of the developmental steps of this soft-tissue sarcoma. By investigation of several tumors of the same patient, a number of corresponding changes were always detected. Adult fibrosarcomas from patients who died during the observation time showed statistically significant more frequent gains of 8q, 12q, 13q, and 15q compared to the fibrosarcomas of patients who are alive. Gains and high-level amplifications of 12q14-q22, which were the most frequent genomic imbalances, partly reflected an MDM2 amplification, indicating the importance of this region in the tumorigenesis of sarcomas. In adult fibrosarcomas, a gain of 12q22 correlated significantly (P = 0.028) with a poor overall survival rate.

Increased survivin transcript levels: an independent negative predictor of survival in soft tissue sarcoma patients.

Survivin, a recently identified inhibitor of apoptosis protein (IAP), is expressed in diverse embryonic tissues and in various human cancers. We have investigated the quantitative expression of survivin mRNA by a sensitive TaqMan-based RT-PCR assay in tissue samples from 94 patients with soft tissue sarcomas (STS). Survivin transcript levels were measured and normalized to GAPDH transcripts. By using a multivariate Cox regression analysis, we found an inverse correlation between the level of survivin mRNA (ratio >2 zmol survivin/amol GAPDH) and the rate of overall survival (p = 0.009, RR = 2.7). Survivin transcript variants as detected by qualitative RT-PCR analysis were revealed in 36 of 56 STS patients (64%). Only survivin DeltaEx3 and/or full-length survivin variants but not survivin 2B were identified. Our results suggest that a higher level of survivin mRNA is an independent predictor of survival for STS patients.

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.

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.

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.

Amplification of the MDM2 gene, but not expression of splice variants of MDM2 MRNA, is associated with prognosis in soft tissue sarcoma.

The MDM2 gene encodes a 90-kDa oncoprotein that is overexpressed in several human carcinomas, osteosarcomas, gliomas and soft tissue sarcomas (STSs). This overexpression is the result of several mechanisms, for example, enhanced transcription or translation, gene amplification and alternative splicing. We found that 19 of 67 (28.4%) STS specimens contained an amplified MDM2 gene. The amplification was more likely to be present in grade 1 tumors than in grade 2 or 3 tumors (58% of grade 1 tumors vs. 15% of grade 2 or 3 tumors, p = 0.001, chi(2) test). Furthermore, patients with tumors that contained an amplified MDM2 gene had a survival estimate (87 months) that was longer than that of patients with tumors that lacked an amplified gene (40 months; p = 0.02, log-rank test). Alternatively and aberrantly spliced MDM2 mRNAs were detected in human STSs by a highly sensitive reverse transcription-polymerase chain reaction method. Of 71 tumor samples, 38 (54%) showed evidence of the spliced forms, which included MDM2-A, MDM2-B and several variants exclusively expressed in STSs. A common feature of all forms was the absence of the MDM2 N-terminal region, which includes the TP53-binding region. Furthermore, the presence of the spliced forms was associated with elevated levels of TP53 (p = 0.01, chi(2) test). Although the presence of spliced forms was associated with late-stage tumor phenotypes (p = 0.05, chi(2) test), we observed no relationship between the presence of splice variants and patient outcome.

Loss of G2/M arrest correlates with radiosensitization in two human sarcoma cell lines with mutant p53.

We have examined the modulation of radiosensitivity by using caffeine in two human sarcoma cell lines both with a p53 mutation (US8-93 and LMS6-93). In both cell lines a strong irradiation-induced G2/M arrest was coupled with a low rate of apoptosis. Incubation with caffeine resulted in a low percentage of S and G2/M cells, associated with an accumulation in G1. With a higher caffeine concentration, we detected a lower clonogenic survival with IC(50) at 2 mM. In both cell lines incubation with caffeine completely prevents the irradiation-induced G2/M arrest. This was connected to radiosensitization, but without direct correlation to an induction of apoptosis. The effect of radiosensitization rose with higher irradiation doses. However, in comparison with LMS6-93, it was stronger in cell line US8-93. A higher radiosensitization in US8-93 correlated with the prevention of strong irradiation-induced G2/M response and higher initial DNA damage. Results of Western hybridization reveal a p53-independent mechanism of radiosensitization caused by caffeine. Our findings suggest that modulation in G2/M regulation may affect a common checkpoint for tumor cells with defective p53 function. Furthermore, our results show that the enhancer effect of caffeine is dependent on a strong reduction in the number of G2/M arrested cells and on an inhibition of DNA damage repair after irradiation.