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Biomedical subjects

Katja Specht

Publications and source records attributed to Katja Specht.

16 recordsLinked to original sources
PubMed2006

Multicenter validation of a gene expression-based prognostic signature in lymph node-negative primary breast cancer.

PURPOSE: We previously identified in a single-center study a 76-gene prognostic signature for lymph node-negative (LNN) breast cancer patients. The aim of this study was to validate this gene signature in an independent more diverse population of LNN patients from multiple institutions. PATIENTS AND METHODS: Using custom-designed DNA chips we analyzed the expression of the 76 genes in RNA of frozen tumor samples from 180 LNN patients who did not receive adjuvant systemic treatment. RESULTS: In this independent validation, the 76-gene signature was highly informative in identifying patients with distant metastasis within 5 years (hazard ratio, [HR], 7.41; 95% CI, 2.63 to 20.9), even when corrected for traditional prognostic factors in multivariate analysis (HR, 11.36; 95% CI, 2.67 to 48.4). The actuarial 5- and 10-year distant metastasis-free survival were 96% (95% CI, 89% to 99%) and 94% (95% CI, 83% to 98%), respectively, for the good profile group and 74% (95% CI, 64% to 81%) and 65% (53% to 74%), respectively for the poor profile group. The sensitivity for 5-yr distant metastasis-free survival was 90%, and the specificity was 50%. The positive and negative predictive values were 38% (95% CI, 29% to 47%) and 94% (95% CI, 86% to 97%), respectively. The 76-gene signature was confirmed as a strong prognostic factor in subgroups of estrogen receptor-positive patients, pre- and postmenopausal patients, and patients with tumor sizes 20 mm or smaller. The subgroup of patients with estrogen receptor-negative tumors was considered too small to perform a separate analysis. CONCLUSION: Our data provide a strong methodologic and clinical multicenter validation of the predefined prognostic 76-gene signature in LNN breast cancer patients.

Adult↗
PubMed2005

Ancillary techniques in bone marrow pathology: molecular diagnostics on bone marrow trephine biopsies.

Pathologic examination of trephine bone marrow (BM) biopsies plays a central role in the diagnosis and staging of haematological neoplasms and other disorders affecting haematopoiesis. Haematopathology has been profoundly influenced by the advent of molecular genetic techniques suitable for paraffin-embedded tissues, and certain applications, such as the determination of B- and T-cell clonality, belong to its standard diagnostic repertoire. Many of these molecular tests can be performed successfully with nucleic acids extracted from BM trephine biopsies, if some technical aspects specific to this template source such as various fixation and decalcification procedures are taken into consideration. The current indications for molecular BM diagnostics range from the confirmation of lymphoma involvement with gene rearrangement analysis, demonstration of tumor-specific translocations in lymphoid and chronic myeloproliferative disorders along to the detection of microorganisms or marrow involvement by soft tissue sarcomas. The availability of quantitative polymerase chain reaction techniques for the investigation of allelic imbalances and gene expression levels in paraffin-embedded material also open new avenues for research and advanced diagnostics. The molecular detection of minimal residual disease in haematological neoplasms, especially in the context of new treatment strategies, will provide future challenges. This article summarizes the current state of the art in molecular diagnostics applied to paraffin-embedded BM biopsies.

Biopsy↗
PubMed2005

Association of pretherapeutic expression of chemotherapy-related genes with response to neoadjuvant chemotherapy in Barrett carcinoma.

PURPOSE: We analyzed pretherapeutic gene expression patterns of patients with locally advanced adenocarcinomas of the esophagus with regard to response to neoadjuvant chemotherapy. EXPERIMENTAL DESIGN: Pretherapeutic, paraffin-embedded, formalin-fixed endoscopic esophageal tumor biopsies of 38 patients with locally advanced esophageal adenocarcinomas (Barrett adenocarcinoma) were included. All patients underwent two cycles of cisplatin and 5-fluorouracil (5-FU) therapy with or without additional paclitaxel followed by abdominothoracal esophagectomy. RNA expression levels of 5-FU metabolism-associated genes thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, methylenetetrahydrofolate reductase, MAP7, and ELF3, of platinum- and taxane-related genes caldesmon, ERCC1, ERCC4, HER-2/neu, and GADD45, and of multidrug resistance gene MRP1 were determined using real-time reverse transcriptase-PCR. Expression levels were correlated with response to chemotherapy, histopathologically assessed in surgically resected specimens. RESULTS: Responding patients showed significantly higher pretherapeutic expression levels of MTHFR (P = 0.012), caldesmon (P = 0.016), and MRP1 (P = 0.007). In addition, patients with high pretherapeutic MTHFR and MRP1 levels had a survival benefit after surgery (P = 0.013 and P = 0.015, respectively). Additionally, investigation of intratumoral heterogeneity of gene expression of relevant genes (MTHFR, caldesmon, HER-2/neu, ERCC4, and MRP1), verified in nine untreated Barrett adenocarcinomas by examination of five distinct tumor areas, revealed no significant heterogeneity in gene expression indicating that expression profiles obtained from biopsy material may yield a representative genetic expression profile of total tumor tissue. CONCLUSIONS: Our results indicate that determination of mRNA levels of few genes may be useful for the prediction of the success of neoadjuvant chemotherapy in individual cancer patients with locally advanced Barrett adenocarcinoma.

Adenocarcinoma↗
PubMed2005

Combined GADD45A and thymidine phosphorylase expression levels predict response and survival of neoadjuvant-treated gastric cancer patients.

PURPOSE: We evaluated the expression of seven therapy-related genes to predict the clinical outcome of advanced gastric cancer patients treated with a neoadjuvant chemotherapeutic protocol. EXPERIMENTAL DESIGN: Pretherapeutic, formalin-fixed, and paraffin-embedded biopsies of 61 patients, who received a 5-fluorouracil (5-FU)- and cisplatin-based chemotherapy were studied. The expressions of the 5-FU-related genes TS, DPD, and TP and of the cisplatin-related genes ERCC1, ERCC4, KU80, and GADD45A were analyzed by quantitative real-time PCR. The expression levels of single genes and of various combinations were tested for an association with response and overall survival. RESULTS: High DPD levels were more frequently found in nonresponding patients and were associated with worse survival. GADD45A and TP levels showed weak associations with response, but GADD45A expression correlated with survival. There was no association with response for TS expression, but tumors with a high TS level were associated with worse survival. The combination of GADD45A and TP revealed the strongest predictive effect. High expression values of TP and/or GADD45A were exclusively found in nonresponding patients (P = 0.002) and were associated with a significantly poorer survival (P = 0.04). CONCLUSIONS: Combined gene expression levels of TP and GADD45A represent a new variable to predict the clinical outcome after neoadjuvant chemotherapy in gastric cancer. The association of DPD expression with response and survival underlines a predominant role of DPD to predict 5-FU sensitivity. The association of TS expression levels with survival but not with response suggests an importance of this gene for tumor progression.

Adult↗
PubMed2005

Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3.

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread.

Biological Assay↗
PubMed2005

Primary extramedullary plasmacytoma and multiple myeloma: phenotypic differences revealed by immunohistochemical analysis.

Primary extramedullary plasmacytomas are infrequent, typically solitary, plasma cell neoplasms that generally pursue an indolent clinical course but may, rarely, convert to multiple myeloma. Phenotypic differences between these two entities are not well defined. Twenty-eight cases of primary extramedullary plasmacytoma and 26 cases of both medullary (n = 17) and extramedullary (n = 9) multiple myeloma were analysed for the expression of proteins known to play a role in the biology of multiple myeloma. Immunohistochemistry was performed on paraffin wax sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, p27, p21, p53, MIB1, CD20, and CD56. Twenty-three extramedullary plasmacytomas were localized in the upper aerodigestive tract, four in the lymph nodes, and one in the testis. There was a strong male predominance (M : F = 6 : 1). None of the patients died from the disease or progressed to multiple myeloma (mean follow-up 50 months). Nine patients developed local relapse and one patient's tumour evolved into a B-cell non-Hodgkin's lymphoma. In contrast to both intra- and extra-medullary multiple myeloma, extramedullary plasmacytoma showed absence of cyclin D1 (p < 0.001) and infrequent expression of CD56 (p < 0.001). Furthermore, extramedullary plasmacytomas were characterized by weaker staining for Bcl-2 protein and rare overexpression of p21 and p53. In comparison to extramedullary multiple myeloma, extramedullary plasmacytoma showed a more mature morphology and lower proliferation indices (p = 0.008). There was no association between the phenotypic parameters investigated and clinical outcome in extramedullary plasmacytoma. In summary, extramedullary plasmacytoma and multiple myeloma show significant immunophenotypic differences, some of which may be of both diagnostic utility and biological relevance.

Adult↗
PubMed2004

Coamplification and coexpression of GRB7 and ERBB2 is found in high grade intraepithelial neoplasia and in invasive Barrett's carcinoma.

The human growth factor receptor-bound protein 7 gene (GRB7), the founding member of a family of adaptor molecules has been shown to regulate cell migration and has been implicated in tumor progression. GRB7 is localized in close proximity to the ERBB2 gene within an amplicon previously identified in Barrett's adenocarcinoma (BCA). We evaluated gene amplification and mRNA expression of GRB7 and ERBB2 in Barrett's carcinoma and its associated precursor lesions to assess their possible role in Barrett's malignant transformation. Copy number and expression levels were analyzed by Q-PCR (GRB7, ERBB2) and QRT-PCR (GRB7, ERBB2) using TaqMan technology and fluorescence in situ hybridization (ERBB2) in a series of 24 laser-microdissected samples of Barrett's carcinomas and 32 samples of associated premalignant lesions. Parallel analysis of gene copy number changes and expression levels demonstrated that GRB7 and ERBB2 displayed concomitant elevated expression levels and increased copy numbers in 32% of Barrett's carcinomas. There was a correlation between GRB7 and ERBB2 in BCA at the DNA level (r(s) = 0.76, p < 0.001) and the mRNA level (r(s) = 0.89, p < 0.001). Moreover, coamplification (r(s) = 0.97, p < 0.001) and coexpression (r(s) = 0.81, p < 0.001) of GRB7 and ERBB2 are shown to be already present in a subset of BCA associated high-grade intraepithelial neoplasia (HGIN), possibly reflecting a role of a concerted expression of GRB7 and ERBB2. No alterations, neither gene amplification nor overexpression were detected in Barrett's carcinoma associated low grade intraepithelial neoplasia (LGIN), intestinal metaplasia (IM) and squamous epithelium, indicating that alterations of GRB7 and ERBB2 are late events in the carcinogenesis of Barrett's esophagus. These findings may be of particular interest because the transition from high grade intraepithelial neoplasia to invasive carcinoma is a crucial step in malignant transformation in Barrett's carcinogenesis and might underline the putative role of GRB7 and ERBB2 in cell migration and tumor invasion.

Adenocarcinoma↗
PubMed2004

Different mechanisms of cyclin D1 overexpression in multiple myeloma revealed by fluorescence in situ hybridization and quantitative analysis of mRNA levels.

The t(11;14)(q13;q32) is the most common translocation in multiple myeloma (MM), resulting in up-regulation of cyclin D1. We used a segregation fluorescence in situ hybridization (FISH) assay to detect t(11;14) breakpoints in primary MM cases and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify cyclin D1 and MYEOV (myeloma overexpressed) expression, another putative oncogene located on chromosome 11q13. High levels of cyclin D1 mRNA (cyclin D1/TBP [TATA box binding protein] ratio > 95) were found exclusively in the presence of a t(11;14) translocation (11/48 cases; P <.00001). In addition, a subgroup of MM cases (15/48) with intermediate to low cyclin D1 mRNA (cyclin D1/TBP ratio between 2.3 and 20) was identified. FISH analysis ruled out a t(11; 14) translocation and 11q13 amplification in these cases; however, in 13 of 15 patients a chromosome 11 polysomy was demonstrated (P <.0001). These results indicate an effect of gene dosage as an alternative mechanism of cyclin D1 deregulation in MM. The absence of chromosome 11 abnormalities in 2 of 15 patients with intermediate cyclin D1 expression supports that there are presumably other mechanism(s) of cyclin D1 deregulation in MM patients. Our data indicate that deregulation of MYEOV is not favored in MM and further strengthens the role of cyclin D1 overexpression in lymphoid malignancies with a t(11;14)(q13;q32) translocation.

Chromosomes, Human, Pair 11↗
PubMed2004

Strategies to overcome resistance to targeted protein kinase inhibitors.

Selective inhibition of protein tyrosine kinases is gaining importance as an effective therapeutic approach for the treatment of a wide range of human cancers. However, as extensively documented for the BCR-ABL oncogene in imatinib-treated leukaemia patients, clinical resistance caused by mutations in the targeted oncogene has been observed. Here, we look at how structural and mechanistic insights from imatinib-insensitive Bcr-Abl have been exploited to identify second-generation drugs that override acquired target resistance. These insights have created a rationale for the development of either multi-targeted protein kinase inhibitors or cocktails of selective antagonists as antitumour drugs that combine increased therapeutic potency with a reduced risk of the emergence of molecular resistance.

Amino Acid Sequence↗
PubMed2003

Molecular analysis of gene expression in tumor pathology.

Human cancers are diverse in their pathology and responsiveness to clinical treatment. This diversity is at least in part due to variations in cellular gene expression programs. Although the analyis of proteins--the key players in cells and potential drug targets--is advancing rapidly, there are situations in which the analysis of RNA rather than proteins can provide valuable information for the diagnosis of cancer. These situations include absense of an antibody for the protein of interest, expression of functionally defective proteins, expressed small nucleotide polymorphisms (SNPs), analysis of alternatively or abnormally spliced molecules, and functional analysis of splice site mutations. In this chapter we will focus on the analysis of RNA from clinical samples and will summarize how gene expression studies on the RNA level using a variety of new tools can be useful for discovering new classes of tumors, for predicting clinical outcome or therapy response, and for designing novel personalized clinical interventions that can not be achieved with histology alone.

Cadherins↗
PubMed2003

Analysis of signal transducer and activator of transcription 3 (Stat 3) pathway in multiple myeloma: Stat 3 activation and cyclin D1 dysregulation are mutually exclusive events.

The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activation are, mutually exclusive events in MM (P = 0.0066). The universal expression of Mcl-1, independent of activated Stat 3, suggests that its expression is constitutive and that it might play an important role in the pathogenesis of MM.

Aged↗
PubMed2002

Cancer progression and tumor cell motility are associated with the FGFR4 Arg(388) allele.

Expression analysis of genes encoding components of the phosphotyrosine signaling system by cDNA array hybridization revealed elevated levels of FGFR4 transcripts in several mammary carcinoma cell lines. In the FGFR4 gene transcript from MDA-MB-453 mammary carcinoma cells, a G to A conversion was discovered that results in the substitution of glycine by arginine at position 388 in the transmembrane domain of the receptor. The Arg(388) allele was also found in cell lines derived from a variety of other tumor types as well as in the germ-line of cancer patients and healthy individuals. Analysis of three geographically separated groups indicated that it occurs in approximately 50% of the human population. Investigation of the clinical data of 84 breast cancer patients revealed that homo- or heterozygous carriers of the Arg(388) allele had a significantly reduced disease-free survival time (P = 0.01) within a median follow-up of 62 months. Moreover, the FGFR4 Arg(388) allele was associated with early lymph node metastasis and advanced tumor-node-metastasis (TNM) stage in 82 colon cancer patients. Consistent with this finding, MDA-MB-231 mammary tumor cells expressing FGFR4 Arg(388) exhibited increased motility relative to cells expressing the FGFR4 Gly(388) isotype. Our results support the conclusion that the FGFR4 Arg(388) allele represents a determinant that is innocuous in healthy individuals but predisposes cancer patients for significantly accelerated disease progression.

Alleles↗
PubMed2002

Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer.

Epithelial-mesenchymal transition (EMT) involving down-regulation of E-cadherin is thought to play a fundamental role during early steps of invasion and metastasis of carcinoma cells. The aim of our study was to elucidate the role of EMT regulators Snail, SIP1 (both are direct repressors of E-cadherin), and Twist (an activator of N-cadherin during Drosophila embryogenesis), in primary human gastric cancers. Expression of Snail, SIP1, and Twist was analyzed in 48 gastric carcinomas by real-time quantitative RT-PCR in paraffin-embedded and formalin-fixed tissues. The changes of expression levels of these genes in malignant tissues compared to matched non-tumorous tissues were correlated with the expression of E- and N-cadherin. From 28 diffuse-type gastric carcinomas analyzed reduced E-cadherin expression was detected in 11 (39%) cases compared to non-tumorous tissues. Up-regulated Snail could be found in 6 cases with reduced or negative E-cadherin expression. However, there was no correlation to increased SIP1 expression. Interestingly, we could detect abnormal expression of N-cadherin mRNA in 6 cases, which was correlated with Twist overexpression in 4 cases. From 20 intestinal-type gastric cancer samples reduced E-cadherin expression was found in 12 (60%) cases, which was correlated to up-regulation of SIP1, since 10 of these 12 cases showed elevated mRNA levels, whereas Snail, Twist, and N-cadherin were not up-regulated. We present the first study investigating the role of EMT regulators in human gastric cancer and provide evidence that an increase in Snail mRNA expression is associated with down-regulation of E-cadherin in diffuse-type gastric cancer. We detected abnormally positive or increased N-cadherin mRNA levels in the same tumors, probably due to overexpression of Twist. SIP1 overexpression could not be linked to down-regulated E-cadherin in diffuse-type tumors, but was found to be involved in the pathogenesis of intestinal-type gastric carcinoma. We conclude that EMT regulators play different roles in gastric carcinogenesis depending on the histological subtype.

Cadherins↗
PubMed2002

Identification of cyclin D1 mRNA overexpression in B-cell neoplasias by real-time reverse transcription-PCR of microdissected paraffin sections.

PURPOSE: Overexpression of cyclin D1 mRNA and protein as a result of the chromosomal translocation t(11;14)(q13;q32) is a highly specific molecular marker of mantle cell lymphoma, but cyclin D1 dysregulation can also be found in other B-cell neoplasias. The aim of the study was to develop a precise and reliable tool for quantitation of cyclin D1 mRNA suitable for archival clinical specimens. EXPERIMENTAL DESIGN: A real-time reverse transcription-PCR (RT-PCR) assay was used to quantitate cyclin D1 mRNA copy numbers. Using 2000 microdissected cells as template, 104 formalin-fixed, paraffin-embedded lymph node, spleen, and decalcified bone marrow biopsies from a panel of 95 cases of B-cell non-Hodgkin's lymphomas (B-NHLs) were analyzed. In addition, cyclin D1 protein expression was assessed by immunohistochemistry. RESULTS: Strong cyclin D1 mRNA overexpression was detected in mantle cell lymphomas (23 of 23), hairy cell leukemias (5 of 19), and multiple myelomas (7 of 23) with particularly high levels in 2 of the latter cases. Intermediate transcript levels were found in 5 of 23 multiple myelomas and 7 of 19 hairy cell leukemias. B-cell chronic lymphocytic leukemias (10 of 10), follicular lymphomas (9 of 9), mucosa-associated lymphoid tissue lymphomas (5 of 5) and reactive lymphoid tissues with the exception of normal spleen had no or very low cyclin D1 expression. In comparison with real-time RT-PCR, immunohistochemistry showed a lower level of sensitivity, more variability, and did not allow accurate quantitation. CONCLUSIONS: Real-time RT-PCR for cyclin D1 mRNA is an excellent tool for the differential diagnosis of B-NHLs and, in combination with microdissection, a powerful approach for retrospective trials using archival clinical specimens as tissue source. Furthermore, real-time RT-PCR may help to identify subgroups of B-NHLs according to cyclin D1 mRNA copy numbers and to investigate the possible influence of different chromosomal breakpoints on cyclin D1 expression.

Chromosomes, Human, Pair 11↗