Biomedical subjects

T Pietsch

Publications and source records attributed to T Pietsch.

At least 19 recordsLinked to original sources

PubMed2002

Human medulloblastomas lack point mutations and homozygous deletions of the hSNF5/INI1 tumour suppressor gene.

Medulloblastomas (MBs) are malignant primitive neuroectodermal tumours (PNETs) of the cerebellum occurring predominantly in childhood. The association of monosomy of chromosome 22 with MB is controversial. Atypical teratoid/rhabdoid tumours (AT/RTs) of the brain share clinical and histological features with MBs and supratentorial PNETs (sPNETs). In particular, AT/RTs can be misdiagnosed as MBs and sPNETs because AT/RTs frequently contain areas of primitive neuroepithelial cells similar to PNETs. Recently, mutations of the tumour suppressor gene hSNF5/INI1, located on 22q11.23, have been described in AT/RTs, MBs and sPNETs, with conflicting data on the prevalence of hSNF5/INI1 mutations in the latter entities. Therefore, we screened MBs for point mutations and homozygous deletions of the hSNF5/INI1 tumour suppressor gene. In 90 MBs, no mutations of the hSNF5/INI1 gene were identified. Thus, our study virtually rules out hSNF5/INI1 as a tumour suppressor gene involved in the pathogenesis of medulloblastoma.

Adolescent↗

PubMed2002

Molecular genetic analysis of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 tumour-associated genes in supratentorial primitive neuroectodermal tumours and glioblastomas of childhood.

Supratentorial primitive neuroectodermal tumours (sPNETs) are malignant central nervous system tumours of childhood which are histologically characterized by poorly differentiated neuroepithelial cells with the capacity for divergent differentiation into glial, neuronal, myogenic or melanotic lines. The histological differential diagnosis between sPNET and glioblastoma multiforme (GBM) may be difficult, particularly as GBMs can sometimes demonstrate a poorly differentiated PNET-like phenotype. To identify molecular genetic markers that may distinguish sPNET and GBM, we investigated 12 cerebral sPNETs and six GBMs from paediatric patients for genetic alterations of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 genes, as well as for allelic loss on chromosome arms 10q and 17p. Mutations of the TP53 tumour suppressor gene were found in one of 12 sPNETs (8%) and two of six GBMs (33%). None of the sPNETs but two of six GBMs (33%, including one GBM with a TP53 mutation) showed allelic losses on chromosome arm 17p. PTEN mutations were detected in one of 12 sPNET (8%) and one of six GBMs (17%). None of the sPNETs and GBMs carried a homozygous deletion involving the CDKN2A tumour suppressor gene. No amplification of the EGFR, CDK4 or MDM2 proto-oncogenes was detected. Taken together, our results indicate that paediatric GBMs differ from sPNETs by a higher incidence of allelic losses on 17p and TP53 mutations. In addition, the patterns of genetic alterations in sPNETs and paediatric GBMs appear to be distinct from those in cerebellar medulloblastomas and adult GBMs, respectively.

Adolescent↗

PubMed2001

Methylation changes in the human IGF2 p3 promoter parallel IGF2 expression in the primary tumor, established cell line, and xenograft of a human hepatoblastoma.

Hepatoblastoma (HB) is a rare malignant embryonal liver tumor. Its pathogenesis has been associated with altered regulation of the IGF2 and H19 genes, and previous studies have suggested a correlation between abnormal methylation and altered expression of these genes in hepatoblastoma. Upregulation of the activity of the IGF2 promoter P3 has previously been shown to be tightly correlated with demethylation in hepatoblastoma. Here, we have used bisulfite genomic sequencing to characterize the methylation pattern of the IGF2 promoter P3 in the hepatoblastoma-derived cell line Hep T1, in the original tumor from which Hep T1 is derived, and in nude mouse xenografts of the Hep T1 cell line. The results show a clear difference in methylation pattern of the most proximal region of the IGF2 P3 promoter between the primary tumor, the cell line, and the xenografts. RNase protection and mRNA in situ hybridization revealed that variations in methylation patterns was paralleled by the levels of IGF2 P3 mRNA, which was detectable in the primary tumor and xenografts, but not in the cell line. Furthermore, it was demonstrated that H19 was reactivated and demethylated in the HepT1 cell line by 5-azaCytidine, in contrast to IGF2 P3, which was not demethylated or reactivated. We suggest that methylation of the proximal IGF2 P3 is important for its regulation.

Animals↗

PubMed2001

Deletions of AXIN1, a component of the WNT/wingless pathway, in sporadic medulloblastomas.

Medulloblastoma (MB) represents the most frequent malignant brain tumor in children. Most MBs appear sporadically; however, their incidence is highly elevated in two inherited tumor predisposition syndromes, Gorlin's and Turcot's syndrome. The genetic defects responsible for these diseases have been identified. Whereas Gorlin's syndrome patients carry germ-line mutations in the patched (PTCH) gene, Turcot's syndrome patients with MBs carry germ-line mutations of the adenomatous polyposis coli (APC) gene. The APC gene product is a component of a multiprotein complex controlling beta-catenin degradation. In this complex, Axin plays a major role as scaffold protein. Whereas APC mutations are rare in sporadic MBs, a hot-spot region of beta-catenin (CTNNB1) mutations was identified in a subset of MBs. To find out if Axin is also involved in the pathogenesis of sporadic MBs, we analyzed 86 MBs and 11 MB cell lines for mutations in the AXIN1 gene. Using single-strand conformation polymorphism analysis, screening for large deletions by reverse transcription-PCR, and sequencing analysis, a single somatic point mutation in exon 1 (Pro255Ser) and seven large deletions (12%) of AXIN1 were detected. This indicates that AXIN1 may function as a tumor suppressor gene in MBs.

Adolescent↗

PubMed2001

Somatic mutations of WNT/wingless signaling pathway components in primitive neuroectodermal tumors.

Primitive neuroectodermal tumors (PNETs) represent the most frequent malignant brain tumors in childhood. The majority of these neoplasms occur in the cerebellum and are classified as medulloblastomas (MB). Most PNETs develop sporadically; however, their incidence is highly elevated in patients carrying germline APC gene mutations. The APC gene encodes a central component of the WNT/wingless developmental signaling pathway. It regulates the levels of cytoplasmic beta-catenin protein that plays a central role in neural development and cell proliferation. We analyzed 87 sporadic PNETs and 10 PNET cell lines for mutations of the APC gene and beta-catenin (CTNNB1) gene using single strand conformational polymorphism (SSCP) and sequencing analysis. We examined the mutation cluster region of APC (codons 1255--1641) for germline variants and somatic mutations. The medulloblastoma cell line MHH-MED-2 carried a Glu1317Gln missense germline variant and a sporadic MB sample showed a somatic Pro1319Leu substitution. Mutational analysis of exon 3 of CTNNB1 uncovered 4 PNETs (4.8%) with somatic missense mutations. These mutations caused amino acid substitutions in 3 of 80 medulloblastomas (Ser33Phe, Ser33Cys and Ser37Cys) and 1 of 4 supratentorial PNETs (Gly34Val). All mutations affected GSK-3 beta phosphorylation sites of the degradation targeting box of beta-catenin and resulted in nuclear beta-catenin protein accumulation. Deletions of CTNNB1 were not detected by PCR amplification with primers spanning exons 1--5. Our data indicate that inappropriate activation of the WNT/wingless signaling pathway by mutations of its components may contribute to the pathogenesis of a subset of PNETs.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Allelic loss at 10q23.3 but lack of mutation of PTEN/MMAC1 in chromophobe renal cell carcinoma.

Chromophobe renal cell carcinoma (RCC) is characterized by loss of multiple chromosomes including chromosome 10. This study was undertaken to determine the LOH at the PTEN/MMAC1 locus (chromosome band 10q23.3) and to search for gene mutations in 15 chromophobe, 50 conventional, and 10 papillary RCCs as well as in 10 renal oncocytomas. Loss of heterozygosity (LOH) wa seen at all informative loci in all chromophobe RCCs and in two conventional RCCs. We did not find mutations by analyzing exon 1 to 9 of the PTEN/MMAC1 gene using the PCR-SSCP technique in tumors with LOH at 10q23.3.

Carcinoma, Renal Cell↗

PubMed2001

Identification of amplified genes from SV40 large T antigen-induced rat PNET cell lines by subtractive cDNA analysis and radiation hybrid mapping.

Primitive neuroectodermal tumors (PNETs) such as human medulloblastomas are genetically heterogeneous and therefore poorly understood. In a rat model the SV40 large T antigen was used to induce neoplasms with characteristic features of PNETs. Tumor development requires a latency period of 8-11 months implicating secondary genetic alterations. To identify such secondary alterations we performed comparative analyses of two phenotypically identical PNET-derived cell lines. Indeed, these cell lines displayed distinct high-level amplification sites. Using a combination of subtractive cDNA analysis and radiation hybrid mapping we have now identified genes in the amplicon regions of the two cell lines. Interestingly, one of these genes encodes the rat homolog of a cytosolic branched chain aminotransferase (BCAT(C)) previously shown to be amplified in a mouse teratocarcinoma cell line. We propose that this simple cloning strategy may serve as a powerful tool for the isolation of genes implicated in known chromosomal aberrations in primary tumors and tumor cell lines.

Amino Acid Sequence↗

PubMed2001

Novel ERBB4 juxtamembrane splice variants are frequently expressed in childhood medulloblastoma.

We recently reported a significant relationship between tumor cell expression of the ERBB4 receptor, the most recently described member of the epidermal growth factor receptor family, and aggressive tumor phenotype in childhood medulloblastoma. Two alternative juxtamembrane (JM) isoforms of the ERBB4 receptor have been described. Termed JMa and JMb, these variants possess different receptor processing and ligand-binding characteristics. In the current study, we employed an RT-PCR and sequencing strategy to determine the pattern of ERBB4 JM isoform expression in a large (n = 78) series of pediatric medulloblastomas. JMa and JMb transcript expression was detected in 53% and 28% of tumor samples, respectively. In addition, two novel ERBB4 JM isoforms, which we have termed JMc and JMd, were isolated from 10% and 36% of tumors, respectively. Sequence analysis revealed the JMc transcript to contain a deletion of the entire JM region. In contrast, JMd includes an extended coding region, retaining both the JMa and JMb sequences. Neither of these novel isoforms was detected in normal human adult cerebellum, but expression of JMd was observed in developing fetal cerebellum, suggesting that this later isoform may represent an ERBB4 transcript restricted to primitive neuroectoderm-derived tissue. To confirm that the four ERBB4 JM isoforms arise by alternative RNA splicing, we sequenced the intron-exon junctions of the human ERBB4 gene within the JM region. This demonstrated the four ERBB4 JM variants to be encoded by two short exons containing the JMb and JMa sequences positioned in the order 5' to 3' and separated by a 121 bp intron.

Alternative Splicing↗

PubMed2001

Plasma levels of macrophage migration inhibitory factor are elevated in patients with severe sepsis.

OBJECTIVE: To investigate the role of macrophage migration inhibitory factor (MIF) as a marker of severity of systemic inflammation in patients with severe sepsis and critically ill postsurgical patients. DESIGN: Prospective observational study in consecutive patients with severe sepsis, critically ill nonseptic postsurgical patients, and healthy blood donors. SETTING: A surgical intensive care unit of a university hospital. PATIENTS AND PARTICIPANTS: 19 patients with severe sepsis, 18 critically ill nonseptic postsurgical patients, and 10 healthy blood donors. MEASUREMENTS AND RESULTS: MIF plasma levels of patients and participants were measured. Interleukin 6 plasma levels were monitored as a control marker of inflammation. The median MIF plasma level was four to five times higher in patients with severe sepsis (2.70 ng/ml, range 0.31-19.59) and in critically ill nonseptic postsurgical patients (2.43 ng/ml, range 0.49-4.31) than in healthy blood donors (0.56 ng/ml, range 0.16-1.68). MIF plasma levels did not differ between the patient groups. CONCLUSIONS: MIF serves as a general marker for systemic inflammation in septic and nonseptic acute critical illness, but MIF does not discriminate for severity or differentiate between infectious and noninfectious origins of an acute critical illness.

Adolescent↗

PubMed2001

Indications for a tumor suppressor gene at 22q11 involved in the pathogenesis of ependymal tumors and distinct from hSNF5/INI1.

Ependymomas account for approximately 9% of all neuroepithelial tumors and represent the most frequent neuroepithelial tumors of the spinal cord. In adults, allelic loss of chromosome arm 22q occurs in up to 60% of the cases studied. Some of these tumors show an altered neurofibromatosis type 2 (NF2) gene; in others, NF2 appears to be unaffected, indicating the involvement of another tumor suppressor gene. Recently, the tumor suppressor gene hSNF5/INI1, located on 22q11.23, has been shown to contribute to the pathogenesis of renal and extrarenal rhabdoid tumors. In addition, this gene may be responsible for a new hereditary syndrome predisposing to a variety of tumors designated "rhabdoid predisposition syndrome." In the present study, we analyzed a series of 53 ependymal tumors of 48 patients [4 myxopapillary ependymomas (WHO grade I), 3 subependymomas (WHO grade I), 18 ependymomas (WHO grade II), 21 anaplastic ependymomas (WHO grade III) and 2 ependymoblastomas (WHO grade IV)] for mutations and homozygous deletions in the coding region of the hSNF5/INI1 gene and for allelic loss of its flanking chromosomal regions in 39 ependymal tumors of 35 patients. Allelic loss was detected in 11 of 35 informative primary ependymal tumors (31%) with a common region of overlap covered by the markers D22S257 and D22S310 on 22q11 including the marker D22S301. However, a detailed molecular analysis of 53 ependymal tumors for mutations and homozygous deletion of the hSNF5/INI1 gene revealed no alterations. We conclude that the hSNF5/INI1 gene is not involved in the pathogenesis of human ependymal tumors with allelic loss on chromosome arm 22q and an intact NF2 locus. In addition, our study localizes a putative ependymoma tumor suppressor gene(s) to a domain of chromosome arm 22q flanked by the microsatellite markers D22S257 and D22S310.

Adolescent↗

PubMed2001

Analysis of the TSC2 gene in human medulloblastoma.

Medulloblastoma (MB) represents the most frequent malignant brain tumor of childhood. Recent studies have shown that deregulation of developmental control genes may play an important role in its pathogenesis. Tuberous sclerosis is associated with hamartomas and cortical tubers, consisting of both glial and neuronal cellular components. MBs can also show markers of these lineages, raising the question of the potential involvement of TSC genes in these malignant tumors. Here we investigated tuberous sclerosis 2 (TSC2), one of the two genes responsible for tuberous sclerosis, in sporadic MBs. We analyzed MBs for allelic losses at the TSC2 locus and for the frequency of a polymorphism first described in gangliogliomas. Sixty-eight MBs were examined for this polymorphism located in intron 4, 3 base pairs 5' to the first coding nucleotide of exon 5. The distribution of the alleles was significantly different in MBs as compared to 208 control samples, (P=0.0017, Chi-square test). In MBs the frequency of the rare allele (A2) was 0.184 (18.4%), whereas in the control group it occurred in a frequency of 8.7%. Microsatellite analysis of the TSC2 region in 50 tumors did not identify allelic losses. TSC2 mRNA transcript was detectable via reverse transcription-PCR in all tumors as well as in normal cerebellum. Northern blot analysis of an MB cell line homozygous for the rare allele of the polymorphism and two other cell lines homozygous for the frequent allele revealed normal splicing patterns and normal expression levels of the TSC2 transcript. These findings may indicate that the presence of the rare TSC2 allele is associated with a predisposition for the development of MBs.

Blotting, Northern↗

PubMed2001

New developments in the pathology of skull base tumors.

As an anatomical interface between various tissues, the skull base harbors an exceptionally broad variety of neoplasms, some of which pose a major challenge for surgical pathology. The characterization of distinct immunohistochemical expression profiles and the identification of molecular genetic alterations associated with different tumor entities have significantly advanced this field. The new World Health Organization (WHO) classification of tumors of the nervous system lists 15 histopathological variants of meningioma. Of clinical importance are those entities that carry an increased risk of recurrence and a poor prognosis, i.e., the atypical meningioma (WHO grade II), clear-cell meningioma (WHO grade II), chordoid meningioma (WHO grade II), rhabdoid meningioma (WHO grade III), papillary meningioma (WHO grade III), and anaplastic meningioma (WHO grade III). Diagnostic criteria for atypical and anaplastic meningioma variants have now been stringently defined. The differential diagnosis of meningiomas includes hemangiopericytoma, hemangioblastoma, solitary fibrous tumor, sarcomas, and chordoid neoplasms. Recent data highlight the importance of distinguishing chordoma and chondrosarcoma of the skull base since chondrosarcomas show a significantly better clinical outcome. Among the less common, aggressive tumor entities in this anatomical region, infiltrating pituitary adenoma/pituitary carcinoma, superficial malignant gliomas, rhabdomyosarcoma, olfactory neuroblastoma, various sarcomas, and malignant lymphoma must be considered. Profiles of molecular genetic alterations have been established for several of these neoplasms and may facilitate the differential diagnosis. This review summarizes recent developments in the histopathological characterization, classification, and molecular pathology of neoplasms arising at the skull base.

Biomarkers, Tumor↗

PubMed2001

Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis.

The human insulin-like growth factor-II (IGF2) is a regulatory peptide which is critical in normal fetal growth. IGF2 gene transcription is controlled by the usage of four promoters P1-P4 of which promoters P2-P4 are genomically imprinted. Disruption of imprinting and the resulting increase of gene dosage have been shown to be implicated in tumor progression in a variety of human tumors. Due to the need for high amounts of tissue material for conventional methods such as Northern blotting or ribonuclease protection assay (RPA), studies on IGF2 expression have most often been limited to the detection of total IGF2 transcript, though different dysregulatory events can be responsible for the abundance of IGF2 mRNA found in many tumors. We established a highly sensitive competitive RT-PCR assay for the four different transcripts of the IGF2 gene with transcript-specific external RNA competitors in which we take advantage of fluorescence-based quantification on a semiautomated sequencer. The amount of total RNA needed is approximately 100 times lower than the amounts required for Northern blotting or RPA, so that even cytological samples can be analyzed. We applied the assay to a series of eleven hepatoblastomas (HB) in which normal adjacent liver tissue could also be analyzed.

Child, Preschool↗

PubMed2001

Medulloblastoma displays distinct regional matrix metalloprotease expression.

Matrix metalloproteases (MMPs) play an important role in tissue remodeling and neoangiogenesis during tumor progression. Little is known about the presence and regional distribution of MMPs in medulloblastomas. Based on immunohistochemical, immunocytochemical and zymographical analysis is the present study illustrates the differential pattern of MMP expression for the medulloblastoma compared to that of glioma and ependymoma. In 10 examined medulloblastoma tumors gelatinase-A was strongly expressed by clusters of tumor cells. Gelatinase-B was, similar to glioma and ependymoma, predominantly found around endothelial cells. The DAB signal for macrophage metalloelastase was seen around macrophages and matrilysin was expressed by single tumor cells. Stromelysin-1 protein was detected in medulloblastoma but not in glioma or ependymoma. From the presented data it follows that each tumor entity might display its own characteristic MMP expression pattern.

Astrocytoma↗

PubMed2001

Aberrant hypermethylation of the major breakpoint cluster region in 17p11.2 in medulloblastomas but not supratentorial PNETs.

Deletions of 17p have been consistently reported in up to 50% of medulloblastomas (MBs), and the major breakpoint interval has been localized to chromosome segment 17p11.2. Based on several reports linking aberrant DNA methylation and chromosomal disruption, we examined the methylation pattern in this region by employing restriction landmark genomic scanning (RLGS). Several CpG islands located in the major breakpoint cluster region were identified using a bacterial artificial chromosome (BAC) contig of the breakpoint region. A long-range methylation map was established for 20 MBs and 5 supratentorial primitive neuroectodermal tumors (stPNETs). Selected CpG islands were examined using Southern and bisulfite sequencing analysis. Aberrantly hypermethylated CpG islands in 17p11. 2 were found in 33% of MBs. Interestingly, one CpG island was methylated in MBs, but not in any of the examined stPNETs. A BAC clone covering three of the methylated CpG islands was partially sequenced in the search for a potential tumor suppressor gene. None of the expressed sequence tag sequences and full-length mouse/human cDNAs that were associated with aberrant methylation showed a change in expression levels due to methylation. The potential link between chromosomal instability in 17p11.2 and hypermethylation in this region is discussed.

Adolescent↗

PubMed2000

Oral trofosfamide and etoposide in pediatric patients with glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme in childhood is rare, and the prognosis for patients with the disease is poor. The Pediatric Oncology Society of the Germanic language group (GPOH) enrolls patients in a series of pilot trials, the first of which is reported here (HIT-GBM-A). METHODS: Twenty-two patients with glioblastoma multiforme, World Health Organization Grade 4, between the ages of 3-15 years (45% male) were enrolled during the period 1995-1997. There were 13 supratentorial tumors, 8 brainstem tumors, and 1 cerebellar tumor. The patients underwent the following procedures: stereotactic biopsy (n = 3 patients), open biopsy (n = 1 patient), partial resection (n = 6 patients), subtotal resection (n = 4 patients), and macroscopic total resection (n = 8 patients). Adjuvant treatment consisted of oral chemotherapy with trofosfamide, 100 mg/m(2), and etoposide, 25 mg/m(2), daily or for 21-day cycles interrupted by 1-week rests. Standard fractionated radiation (54 grays) was started concurrently with the first cycle. RESULTS: The chemotherapy was well tolerated, with no treatment-related deaths and only minor side effects. The responses in 12 evaluable patients after two cycles were as follows: 1 complete response, 1 partial response, 3 patients with stable disease, and 7 patients with progressive disease. The median overall survival was 12 months. The 1-year, 2-year, and 4-year overall survival rates were 52%, 26%, and 22%, respectively, and the event free survival rates were 26%, 22%, and 4%, respectively. None of the four surviving patients (3.2 years, 3.4 years, 4.0 years, and 4.2 years after diagnosis) is event free. Two patients are alive after tumor progression: One patient was diagnosed with a medulloblastoma, and one patient was diagnosed with an osteosarcoma as second malignancies. A control group extracted from the Surveillance, Epidemiology, and End Results data had lower survival rates: the difference between the groups was not statistically significant (P = 0.26). CONCLUSIONS: This chemotherapy will not be used in a randomized trial of patients with glioblastoma; however, it may be evaluated for patients with tumors that have more chemoresponsive histologies.

Administration, Oral↗

PubMed2000

C-MYC expression in medulloblastoma and its prognostic value.

To identify prognostic factors in medulloblastoma, a common malignant brain tumor of childhood, expression of the oncogene c-myc was examined at the mRNA level by in situ hybridization. c-myc mRNA expression was observed in 30 of 72 tumors (42%). The c-myc gene copy number was determined by quantitative PCR from genomic DNA of paraffin-embedded tumors. c-myc gene amplification was present in 5 of 62 cases (8.3%). Therefore, c-myc amplification was obviously not the cause of c-myc mRNA expression in most samples. Kaplan-Meier estimation revealed a significant correlation between c-myc mRNA expression and survival (total mean follow-up 4.6 +/- 3.6 years, log-rank p = 0.02). Multivariate logistic regression analysis including sex, age, histological type, degree of surgical resection and expression of synaptophysin, GFAP and c-myc, was carried out on 54 patients who received both radiotherapy and chemotherapy. The analysis identified expression of c-myc as an independent predictive factor of death from disease.

Adolescent↗

PubMed2000

Voltage- and gamma-aminobutyric acid-activated membrane currents in the human medulloblastoma cell line MHH-MED-3.

The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K(+) currents, gamma-aminobutyric acid(A) receptor-mediated Cl(-) currents and most of them inward rectifier K(+) currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca(2+) channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na(+) channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.

Adolescent↗