PTPIP51 levels in glioblastoma cells depend on inhibition of the EGF-receptor.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is upregulated in glioblastoma multiforme (GBM) and expression levels correlate with the grade of malignancy in gliomas. A similar correlation was reported for its interacting partner 14-3-3ß, which has been shown to facilitate the interaction of PTPIP51 with cRAF (Raf1). Since the interaction of these signalling partners stimulates growth factor signalling downstream of the epidermal growth factor receptor (EGFR), a major drug target in GBM, we here investigated the impact of EGFR inhibition by small molecule inhibitors or monoclonal antibody on PTPIP51. The effect of EGFR inhibition on PTPIP51 mRNA, protein expression and its interaction profile in GBM was analyzed using the U87 cell line as model system. The transferability of the results to in vivo conditions was evaluated in cultured tumour cells from GBM patients. Cells were treated either to the small molecule tyrosine kinase inhibitor of EGFR Gefitinib or the monoclonal antibody Cetuximab in a time and dose dependent manner. Gefitinib treatment decreased the proliferation rate and induced apoptosis in U87 and primary tumour cells. The PTPIP51 interaction profile changed in correlation to the applied Gefitinib. Despite unchanged mRNA levels PTPIP51 protein was reduced. In contrast, treatment with Cetuximab had no effects on PTPIP51 expression. In conclusion, our results demonstrate the impact of EGFR inhibition by Gefitinib on PTPIP51 protein expression, a downstream regulator of MAPK signalling. These data will serve as a basis to unravel the precise role of PTPIP51-mediated signalling in GBM and its potential implications for Gefitinib-mediated therapy in future studies.

[Epithelial neuroendocrine tumors of the upper respiratory tract: New entities, new perspectives]. / Neuroendokrine epitheliale Tumoren des oberen Respirationstrakts : Neue Entitäten, neue Blickwinkel.

Epithelial neuroendocrine tumors of the upper respiratory tract are rare and are classified as typical and atypical carcinoid versus small cell neuroendocrine carcinoma. Furthermore, a giant cell variant of neuroendocrine carcinoma is suggested corresponding to the bronchopulmonary system as well as a recently described subtype of oropharyngeal small cell neuroendocrine carcinoma associated with human papillomavirus. Many arguments relying on clinical as well as on molecular findings indicate that the distinction between carcinoid and poorly differentiated neuroendocrine carcinoma does not only reflect different degrees of differentiation of otherwise related tumors but indicates the existence of substantially different types of neoplasms.

Skin metastases in metastatic uveal melanoma: GNAQ/GNA11 mutational analysis as a valuable tool.

BACKGROUND: Uveal melanomas represent 3.1% of all melanomas, with a high potential of metastatic disease of up to 50%, where the median survival time is 6 months. Though liver metastases dominate as the primary site for metastasis, the existence of primary skin metastases is still under discussion but has been reported in only a few studies. OBJECTIVES: We present two cases in which patients with a known history of uveal melanoma developed melanoma skin metastases. METHODS: Mutational analysis was performed to clarify the origin of the metastases (uvea or skin). RESULTS: The analyses revealed GNA11 mutations, which are typical for uveal melanoma. These cases strongly suggest the skin to be the primary site of uveal melanoma. CONCLUSIONS: Knowledge about the mutational status of uveal melanomas opens the opportunity for future targeted therapies that directly interact with the mutation and its activated signal cascades. First trials in uveal melanoma have shown promising results with MEK inhibitors.

[A 75-year-old female patient with pleural effusion and gastric metastases of a poorly differentiated carcinoma]. / 75-jährige Patientin mit Pleuraerguss und gastralen Metastasen eines wenig differenzierten Karzinoms.

A 75-year-old woman was found to have left-sided pleural effusion and endoscopy revealed the rare entity of adenoid cystic carcinoma metastases in the gastric mucosa. Approximately 20% of patients with this carcinoma suffer from distant metastases. For the initial staging detection of adenoid cystic carcinoma metastasis with positron emission tomography (PET) or PET computed tomography (CT) is recommended. The recurrent t(6;9)(q22-23;p23-24) translocation that results in a fusion of the two transcription factor genes MYB and NFIB is detectable in half of the cases. As in our case molecular pathology can confirm the correct diagnosis and identification of the localization of the primary tumor.

[Significance of frozen section diagnosis for the management of laryngeal tumors]. / Bedeutung der Schnellschnittdiagnostik für das Management laryngealer Tumoren.

The frozen section procedure for immediate intraoperative pathological diagnosis represents a pivotal method in tumor diagnosis. In laryngeal tumors the most frequent indication for the use of this method is the documentation of the residual tumor status, while intraoperative consultation with the purpose of primary tumor diagnosis is less common. The specimen management employed in each case should be chosen depending on the clinical question: while the collection of a maximum amount of tissue is advisable for the determination of the residual tumor status, sparing a portion of the remaining tissue for possible future examinations is advisable in the case of primary tumor diagnosis. Moreover, intraoperative frozen section diagnosis with no immediate consequences should be avoided.

Survival and clonal expansion of mutating "forbidden" (immunoglobulin receptor-deficient) epstein-barr virus-infected b cells in angioimmunoblastic t cell lymphoma.

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a peculiar T cell lymphoma, as expanding B cell clones are often present besides the malignant T cell clones. In addition, large numbers of Epstein-Barr virus (EBV)-infected B cells are frequently observed. To analyze the differentiation status and clonal composition of EBV-harboring B cells in AILD, single EBV-infected cells were micromanipulated from lymph nodes of six patients with frequent EBV(+) cells and their rearranged immunoglobulin (Ig) genes analyzed. Most EBV-infected B cells carried mutated Ig genes, indicating that in AILD, EBV preferentially resides in memory and/or germinal center B cells. EBV(+) B cell clones observed in all six cases ranged from small polyclonal to large monoclonal expansions and often showed ongoing somatic hypermutation while EBV(-) B cells showed little tendency for clonal expansion. Surprisingly, many members of expanding B cell clones had acquired destructive mutations in originally functional V gene rearrangements and showed an unfavorable high load of replacement mutations in the framework regions, indicating that they accumulated mutations over repeated rounds of mutation and division while not being selected through their antigen receptor. This sustained selection-free accumulation of somatic mutations is unique to AILD. Moreover, the survival and clonal expansion of "forbidden" (i.e., Ig-deficient) B cells has not been observed before in vivo and thus represents a novel type of viral latency in the B cell compartment. It is likely the interplay between the microenvironment in AILD lymph nodes and the viral transformation that leads to the survival and clonal expansion of Ig-less B cells.

Rare occurrence of classical Hodgkin's disease as a T cell lymphoma.

Recent work identified Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) as clonal progeny of mature B cells. Therefore, it is generally assumed that cHD homogenously represents a B cell lymphoma. In a subset of cHD, however, H/RS cells expressing T cell-associated proteins may be candidates for alternative lineage derivation. Single H/RS cells with cytotoxic T cell phenotype were micromanipulated from three cases of cHD and analyzed by single cell polymerase chain reaction for immunoglobulin heavy (IgH) and light chain (IgL) gene rearrangements, T cell receptor (TCR)-beta gene rearrangements, and germline configuration of the IgH and TCR-beta loci. H/RS cells from two cases of cHD harbored clonal, somatically mutated Ig gene rearrangements, whereas TCR-beta loci were in germline configuration. In contrast, H/RS cells from an additional case harbored clonal TCR-beta variable/diversity/joining (VDJ) and DJ gene rearrangements, whereas the IgH locus was in germline configuration on both alleles. Thus, in two cases of cHD with H/RS cells expressing cytotoxic T cell molecules, the tumor cells are derived from mature B cells that aberrantly express T cell markers. In a third case, however, H/RS cells were derived from a T cell, demonstrating that cHD can also occur as a T cell lymphoma.

Clonal deleterious mutations in the IkappaBalpha gene in the malignant cells in Hodgkin's lymphoma.

Members of the nuclear factor (NF)-kappaB family of transcription factors play a crucial role in cellular activation, immune responses, and oncogenesis. In most cells, they are kept inactive in the cytosol by complex formation with members of the inhibitor of NF-kappaB (IkappaB) family, whose degradation activates NF-kappaB in response to diverse stimuli. In Hodgkin's lymphoma (HL), high constitutive nuclear activity of NF-kappaB is characteristic of the malignant Hodgkin and Reed-Sternberg (H/RS) cells, which occur at low number in a background of nonneoplastic inflammatory cells. In single H/RS cells micromanipulated from histological sections of HL, we detect clonal deleterious somatic mutations in the IkappaBalpha gene in two of three Epstein-Barr virus (EBV)-negative cases but not in two EBV-positive cases (in which a viral oncogene may account for NF-kappaB activation). There was no evidence for IkappaBalpha mutations in two non-HL entities or in normal germinal center B cells. This study establishes deleterious IkappaBalpha mutations as the first recurrent genetic defect found in H/RS cells, indicating a role of IkappaBalpha defects in the pathogenesis of HL and implying that IkappaBalpha is a tumor suppressor gene.

[Receptor tyrosine kinases in Hodgkin lymphoma as possible therapeutic targets]. / Rezeptor-Tyrosinkinasen in Hodgkin-Lymphomen als mögliche Angriffspunkte neuer Therapieoptionen.

Hodgkin lymphoma (HL) is the most frequent nodal lymphoma in Europe. The B-cell derived Hodgkin-Reed Sternberg (HRS) cells are nearly completely deficient for expression of B-cell markers. Epstein-Barr virus (EBV) can be detected in about 40% of HL cases. Presumably, EBV protects HRS cell precursors from apoptosis. Histologically only single HRS cells are dispersed in a broad reactive cellular background. Interactions between HRS cells and their surrounding cellular infiltrate, among them paracrine activation of several signalling pathways, is crucial in HL. HRS cells also show autocrine activation of several signalling pathways. Among these, the aberrant expression and activation of seven different receptor tyrosine kinases (RTK) is of special interest, as many different antibodies and low molecular substances which inhibit RTK activity are already in clinical use for anticancer therapy. Therefore, blocking of RTK activities in HL may be a novel therapeutic option.

High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma.

Mediastinal large B-cell (MBL) and classical Hodgkin lymphoma (HL) have several pathogenic mechanisms in common. As we recently observed aberrant tyrosine kinase (TK) activities in HL, we now analysed also MBL for such activities. Indeed, MBL and HL were the only B-cell lymphomas where elevated cellular phospho-tyrosine contents were typical features. Three TKs, JAK2, RON and TIE1, not expressed in normal B cells, were each expressed in about 30% of MBL cases, and 75% of cases expressed at least one of the TKs. Among the intracellular pathways frequently triggered by TKs, the PI3K/AKT pathway was activated in about 40% of MBLs and essential for survival of MBL cell lines, whereas the RAF/mitogen-activated protein kinase pathway seemed to be inhibited. No activating mutations were detected in the three TKs in MBL cell lines and primary cases. RON and TIE1 were each also expressed in about 35% and JAK2 in about 53% of HL cases. JAK2 genomic gains are frequent in MBL and HL but we observed no strict correlation of JAK2 genomic status with JAK2 protein expression. In conclusion, aberrant TK activities are a further shared pathogenic mechanism of MBL and HL and may be interesting targets for therapeutic intervention.

Insights into the multistep transformation process of lymphomas: IgH-associated translocations and tumor suppressor gene mutations in clonally related composite Hodgkin's and non-Hodgkin's lymphomas.

Clonally related composite lymphomas of Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL) represent models to study the multistep transformation process in tumorigenesis and the development of two distinct tumors from a shared precursor. We analyzed six such lymphomas for transforming events. The HLs were combined in two cases with follicular lymphoma (FL), and in one case each with B-cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). In the HL/FL and HL/MCL combinations, BCL2/IGH and CCND1/IGH translocations, respectively, were detected in both the HL and NHL. No mutations were found in the tumor suppressor genes FAS, NFKBIA and ATM. The HL/DLBCL case harbored clonal replacement mutations of the TP53 gene on both alleles exclusively in the DLBCL. In conclusion, we present the first examples of molecularly verified IgH-associated translocations in HL, which also show that BCL2/IGH or CCND1/IGH translocations can represent early steps in the pathogenesis of composite HL/FL or HL/MCL. The restriction of the TP53 mutations to the DLBCL in the HL/DLBCL case exemplifies a late transforming event that presumably happened in the germinal center and affected the fate of a common lymphoma precursor cell towards development of a DLBCL.

Somatic mutations of the CD95 gene in Hodgkin and Reed-Sternberg cells.

Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the H/RS cells from apoptosis. Given the importance of CD95 (Apo-1/Fas)-mediated apoptosis for negative selection within the germinal center, single micromanipulated H/RS cells from 10 cases of cHD were analyzed for somatic mutations within the CD95 gene. Three clonal mutations within the 5' regions were amplified from single H/RS cells in one case. From H/RS cells of another case, two mutations within the last exon coding for the death domain were detected. About half of these H/RS cells carried a monoallelic stop-codon; the remaining tumor cells harbored a monoallelic replacement mutation. Both mutations likely impair CD95 function. Because all these H/RS cells also bear clonal mutations inactivating the IkappaB alpha gene, the IkappaB alpha mutations occurred earlier than those of the CD95 gene in the sequence of transforming events leading to cHD. In conclusion, somatic mutations of the CD95 gene occur in a fraction of cHD cases and may favor the escape of the precursor of the H/RS clone from apoptosis.

Identification and functional characterization of the human and murine polo-like kinase (Plk) promoter.

The Plk gene encodes a serine/theronine kinase which is located in the nucleus. Northern blot analysis linked Plk expression to the proliferative activity of cells and tissues. To analyse the transcriptional regulation of the Plk gene we have isolated several human genomic clones containing the Plk promoter. RNAse protection assays revealed three major transcription start sites within a 40 bp region centered around the 5' end of the known human cDNA and 6 minor Cap sites. A genomic fragment of 2.3 kb located 5' to the translation start sites drives the expression of the CAT-reporter in transient transfections in human (EPLC, HeLa) and mouse (NIH3T3, 32D) cell lines in an orientation dependent fashion. The 2.3 kb genomic fragment contains a CCAAT motif located 30-70 bp upstream of the Cap sites and two overlapping Sp1 sites 20 bp further upstream. Additional sequence motif homologues to binding sites of known transcription factors could be identified. In addition to the human Plk promoter, the mouse Plk promoter was isolated. The sequence alignment of the human and murine promoter revealed three regions with extensive sequence homology within a region of 300 bp immediately upstream of the Cap sites. A fourth region of homology encompassing 90 bp about 2.1 kb 5' of the Cap sites was identified as well. Deletion of various regions within the 2.3 kb promoter fragment identified several domains involved in the regulation of the human Plk promoter. The 300 bp region immediately 5' of the Cap sites which is highly conserved between mouse and man is essential for promoter activity. 3' deletions including the CCAAT site abolished promoter activity. Growing 5' deletions within the core region of the promoter reduces transcriptional activity. Furthermore, using deletion clones we identified regions 5' of the core region which enhance or silence the transcriptional activity of the core promoter.

Characterization of the human CSK locus.

The CSK-gene encodes an intracellular protein-tyrosine kinase (PTK). In contrast to members of the src-family, an autophosphorylation site corresponding to Tyr416, as well as the equivalent of the regulatory Tyr527 in p60c-src are missing in the amino acid sequence deduced from the gene. CSK phosphorylates other members of the src-family of tyrosine kinases at their regulatory carboxy-terminus. By regulating the activity of these kinases, CSK may play an important role in cell growth and development. Here we describe the structure of the human CSK gene. The entire coding region spans a genomic distance of only 4.9 kb. It encompasses 12 exons ranging between 66 and 220 bp in size. The introns between coding exons vary between 76 and 920 bp in length. An exon coding for the 5'-untranslated region of CSK is separated from the first coding exon by an intron of more than 6400 bp. Based on comparisons of sequence homologies within the catalytic domains, the intracellular PTKs are divided into the src-family, the fes/fer- and the abl/arg-group. The genomic structure of four members of the SRC-family revealed nearly identical exon/intron boundaries within the catalytic domain of this family. They differ from those described for FES. Comparing the genomic structure of CSK with the exon/intron organisation of both, it is obvious that the exon/intron boundaries are in common either with those of the SRC-type or the FES boundaries. This intermediate exon/intron structure of CSK between FES and the SRC-family agrees with the position of CSK in a phylogenetic tree based on sequence homology within the kinase domain.

Structure, expression and chromosomal mapping of TKT from man and mouse: a new subclass of receptor tyrosine kinases with a factor VIII-like domain.

Using a polymerase chain reaction-mediated approach we have characterized cDNAs from human and mouse origin representing a novel type of receptor protein tyrosine kinase (RTK). The deduced amino acid sequence (855 amino acids) of the longest open reading frame has a unique extracellular region encompassing a factor VIII-like domain, not previously described for RTKs. The most closely related RTKs are members of the neurotrophin receptors (TRK), which showed 47-49% homology with the kinase domain of the new RTK. Therefore, the new gene has been called TKT (Tyrosine-Kinase related to TRK). TKT orthologs from man and mouse were 98% similar. In both species a major transcript of 10 kb was found to be expressed at high levels in heart and lung. Low levels of this mRNA-species were detected in human brain, placenta, liver, skeletal muscle, kidney and in mouse brain and testis. Analysing human/mouse somatic cell hybrids we demonstrated that TKT segregates with human chromosome 1.

Activation induced cytidine deaminase expression in lymphocyte predominant Hodgkin lymphoma.

BACKGROUND: The lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin lymphoma (HL) originate from germinal centre B cells and carry mutated V gene rearrangements, usually with intraclonal diversity. It is unclear whether intraclonal V gene diversification by somatic hypermutation, which is strictly dependent on the enzyme activation induced cytidine deaminase (AID), is restricted to the early phase of lymphoma clone expansion and later silenced, or whether it remains active throughout malignant proliferation. AIMS: To analyse whether AID is expressed in L&H cells as an indicator of active somatic hypermutation in the tumour cells. METHODS: L&H cells from lymphocyte predominant HL cases and centroblasts from lymphadenites were micromanipulated and analysed for AID expression by quantitative real time polymerase chain reaction. RESULTS: The AID transcription level was higher than background in three of the six lymphocyte predominant HL cases, although it was lower than that seen in centroblasts. CONCLUSIONS: Somatic hypermutation may remain active in L&H cells in a considerable proportion of cases, increasing the risk of acquiring further transforming mutations.

Isolation and characterization of a human gene that encodes a new subclass of protein tyrosine kinases.

We describe the isolation and cDNA sequence of a novel human gene, which is distinct from all known members of the human src family of proto-oncogenes. In contrast to these, an autophosphorylation site corresponding to Tyr416, as well as the equivalent of Tyr527 in p60c-src, are missing in the amino acid (aa) sequence deduced from this gene. Furthermore, no N-terminal myristylation site is found. Our human clone is 98% identical at the aa level to a gene which was isolated independently from neonatal rat brain and was termed csk for c-src kinase. We, therefore, propose to designate the present human gene CSK. In Northern blot experiments, CSK was found to be expressed in human lung and macrophages. Due to its extreme conservation across species barriers, the CSK product is likely to exert important regulatory functions. On the basis of its expression in tissues, not typically expressing high c-src levels, it can be assumed that its regulatory role is more general and may also involve other tyrosine kinases.

Oncogenic alterations in primary human lung-tumors (review).

Lung cancer is the leading cause of death from cancer in Western countries. For improved diagnosis and refined therapeutical approaches it is of major importance to understand by what mechanisms carcinoma of the lung develop. The analysis of primary lung cancer revealed specific chromosomal alterations and allelic losses of the short arm of chromosome 3. Genetic aberrations have been observed in proto-oncogenes such as H-ras, K-ras, C-myc and raf-1 as well as in the tumor suppressor genes Rb and p53. Rearrangement of rlf and elevated expression in certain lung tumors have also been reported. The development of lung cancer also involves the altered activation of genes coding for growth factors such as TGF beta 2 and certain growth factor receptor genes such as c-erbB-2, HEK2 and FGFR-4.

Human and Xenopus mo15 messenger-RNA are highly conserved but show different patterns of expression in adult tissues.

Phosphorylation of human p34(cdc2) at Thr 161 seems to be necessary for its catalytic activity. CAK (cdk activating kinase) containing p40(MO15) from Xenopus egg extracts phosphorylates and activates p34(cdc2) in a cyclin dependent manner at Thr 161. We describe the cDNA sequence coding for human MO15, which predicts a serine/threonine kinase of 346 aa. Despite the high homology of 91% between the human and Xenopus proteins we observed a rather different mRNA distribution in adult tissues: In contrast to ubiquitously expressed human MO15-transcripts MO15-mRNA expression in Xenopus is restricted to oocytes indicating a different cellular role in these two phylogenetically distant species. By virtue of the homology to members of the family of cell cycle kinase genes we examined MO15 mRNA expression for its correlation to the proliferative activity of cells. Stimulation of lymphocytes showed MO15 mRNA expression to be independent of mitotic activity.

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.