[Show abstract][Hide abstract] ABSTRACT: National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 suggested the efficacy of adjuvant trastuzumab, even in HER2-negative breast cancer. This finding prompted us to develop a predictive model for degree of benefit from trastuzumab using archived tumor blocks from B-31.
Case subjects with tumor blocks were randomly divided into discovery (n = 588) and confirmation cohorts (n = 991). A predictive model was built from the discovery cohort through gene expression profiling of 462 genes with nCounter assay. A predefined cut point for the predictive model was tested in the confirmation cohort. Gene-by-treatment interaction was tested with Cox models, and correlations between variables were assessed with Spearman correlation. Principal component analysis was performed on the final set of selected genes. All statistical tests were two-sided.
Eight predictive genes associated with HER2 (ERBB2, c17orf37, GRB7) or ER (ESR1, NAT1, GATA3, CA12, IGF1R) were selected for model building. Three-dimensional subset treatment effect pattern plot using two principal components of these genes was used to identify a subset with no benefit from trastuzumab, characterized by intermediate-level ERBB2 and high-level ESR1 mRNA expression. In the confirmation set, the predefined cut points for this model classified patients into three subsets with differential benefit from trastuzumab with hazard ratios of 1.58 (95% confidence interval [CI] = 0.67 to 3.69; P = .29; n = 100), 0.60 (95% CI = 0.41 to 0.89; P = .01; n = 449), and 0.28 (95% CI = 0.20 to 0.41; P < .001; n = 442; P interaction between the model and trastuzumab < .001).
We developed a gene expression-based predictive model for degree of benefit from trastuzumab and demonstrated that HER2-negative tumors belong to the moderate benefit group, thus providing justification for testing trastuzumab in HER2-negative patients (NSABP B-47).
Full-text · Article · Nov 2013 · JNCI Journal of the National Cancer Institute
[Show abstract][Hide abstract] ABSTRACT: Adenocarcinoma in situ (AIS) or high grade cervical glandular intraepithelial neoplasia comprises approximately one percent of cervical in situ lesions and is well accepted as precursor of invasive adenocarcinoma.(1) High-risk human papilloma virus infection has been demonstrated as the most important causative agent of AIS. At present, however, we still know far less about metabolic features of these lesions This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Prevalence and clinical significance of mammalian target of rapamycin (mTOR) phosphorylation at the serine 2448 is disputed in prostate cancer. A tissue microarray containing 3,261 prostate cancers and 49 normal prostate samples with clinical follow-up data was analyzed for p(Ser2448) -mTOR expression by immunohistochemistry. Moderate to strong p(Ser2448) -mTOR staining was found in all (n = 49) normal prostate tissues, but was lost in 24% or weak in 29% cancers. Moderate and strong staining was found in 36 and 11% of tumors. Loss of p(Ser2448) -mTOR staining was significantly linked to advanced stage (p = 0.0027), high-grade (p = 0.0045), nodal positive cancers (p = 0.0483), early tumor recurrence (p < 0.0001, independently from stage and grade, p = 0.0016), lack of Ets-related gene (ERG) fusion (p < 0.0001), reduced androgen receptor expression (p < 0.0001 each) and increased cell proliferation (p = 0.0092) in all cancers and in the subset of ERG-fusion-positive cancers. Loss of p(Ser2448) -mTOR expression was linked to tumor metastasis (p = 0.0275) in ERG-fusion-positive cancers only. Molecular subset analysis using pre-existing phosphatase and tensin homolog (PTEN) deletion data revealed that loss of p(Ser2448) -mTOR expression is of prognostic relevance and defines a subpopulation of PTEN-deleted and ERG-fusion-positive cancers with a particular poor outcome. The results of our study strongly suggest that loss of p(Ser2448) -mTOR expression is a marker for activated AKT/mTOR signaling. Tumors with concomitant PTEN deletion and activated mTOR signaling indicated by loss of p(Ser2448) -mTOR expression characterize a small (4%) but clinically significant subset of prostate cancers that might optimally benefit from anti-mTOR therapies.
Full-text · Article · Mar 2013 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
AIB1 (amplified in breast cancer 1) is an estrogen receptorα (ERα) co-activator, known to be amplified and overexpressed in a fraction of breast cancers. It has been linked to prognosis and tamoxifen resistance. However, results have been ambiguous. The different functions of AIB1 in ERα-positive and -negative disease are poorly understood. Therefore, we analyzed the clinical significance of AIB1 in breast cancer with respect to ERα-status and characterized the subgroups. 2,197 breast carcinomas sampled on a pre-existing tissue microarray (TMA) were analyzed for AIB1 expression and amplification by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).
AIB1 expression was detected in 60 % of the tumors. It was associated with tumor size (p = 0.003), high histological grade (p < 0.0001), poor disease-specific, and overall survival (p = 0.0018 and p = 0.003). There was a strong inverse relationship between AIB1 and ERα expression (p < 0.0001). AIB1 overexpression was associated with increased Ki67 labeling index (p < 0.0001), even if analyzed for different ER expression levels. AIB1 amplification was found in 11 % of the carcinomas. It was associated with high histological grade (p = 0.0012), lymph node involvement (p = 0.0163), and poor disease-specific survival (p = 0.0032) but not with overall survival (p = 0.1672) or ER status (p = 0.4456). If ER-positive tumors were stratified according to their AIB1 amplification status, there was a significant worse disease-specific survival in cases showing AIB1 amplification (p = 0.0017). AIB1 expression is associated with unfavorable prognosis and tumor phenotype. It seems to unfold its oncogenic potential at least in part independent from its role as an ERα co-activator. AIB1 has an impact on cell cycle regulation in ERα-positive as well as ERα-negative tumors. Furthermore, AIB1 amplification characterizes a subgroup of ERα-positive breast cancer with worse outcome. Therefore, AIB1 might be helpful to identify those ERα-positive breast cancers patients who are candidates for adjuvant chemotherapy.
No preview · Article · Jan 2013 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: Next to EGFR mutation, EGFR gene copy number evaluated by fluorescence in situ hybridization (FISH) emerged as a potential predictive marker for sensitivity to EGFR tyrosine kinase inhibitors, although controversial data exist. As the diagnostic accuracy of predictive biomarkers can be substantially limited by regional differences within tumors, heterogeneity of EGFR gene copy gain in NSCLC was assessed in this study. For this purpose, a novel tissue microarray (TMA) based analysis platform was developed. TMAs were constructed containing 8 different tissue cylinders from 144 primary NSCLCs. From 62 of these patients additional nodal metastases were sampled. EGFR gene copy number and EGFR expression was analyzed by FISH and immunohistochemistry according to the suggested guidelines. 13 (9.0%) of the 144 evaluated tumors showed EGFR amplification and 37 (25.7%) tumors high polysomy in at least one tumor area. In 7 (53.8%) of 13 amplified cases the analysis of different tumor areas revealed subclones without EGFR gene copy gain next to subclones with amplification. All of the 36 evaluable tumors with high polysomy showed heterogeneity of EGFR gene copy number with areas negative for gene copy gain within the individual tumors. Heterogeneity of EGFR gene copy gain in lung cancer challenges the concept of using small biopsies for the analysis of EGFR FISH status. EGFR gene copy number is highly heterogeneous within individual NSCLCs and this finding might well be a reason for the controversial clinical data existing regarding responsiveness to anti-EGFR therapy.
No preview · Article · Dec 2012 · Lung cancer (Amsterdam, Netherlands)
[Show abstract][Hide abstract] ABSTRACT: The HER2 protein, encoded by the ERBB2 gene, is a molecular target for the treatment of breast and gastric cancer by monoclonal antibodies or tyrosine kinase inhibitors. While intratumoral heterogeneity of ERBB2 amplification is rare in breast cancer it is reported to be frequent in bladder and colorectal cancer. To address the potential heterogeneity of the HER2 status in adenocarcinomas, squamous cell carcinomas and large cell undifferentiated carcinomas of the lung, 590 tumors were analyzed for HER2 overexpression and ERBB2 amplification using FDA-approved reagents for immunohistochemistry and fluorescence in-situ hybridization (FISH). Moderate and strong immunostaining (2+, 3+) was seen in 10% of the tumors. ERBB2 amplification was found in 17 (3%) lung cancer patients including 10 cases (2%) with high-level amplification forming gene clusters. ERBB2 amplification was significantly related to histologic subtype and tumor grade, resulting in 12% ERBB2 amplified tumors in the subgroup of high-grade adenocarcinomas. Heterogeneity was analyzed in all highly amplified tumors. For this purpose, all available tumor tissue blocks from these patients were evaluated. Heterogeneity of ERBB2 amplification was found in 4 of 10 tumors as assessed by FISH. These included two tumors with a mixture of low-level and high-level amplification and two tumors with non-amplified tumor areas next to regions with high-level ERBB2 amplification. High-level ERBB2 amplification occurs in a small fraction of lung cancers with a strong propensity to high-grade adenocarcinomas. Heterogeneity of amplification may limit the utility of anti-HER2 therapy in some of these tumors. Further attempts to assess the utility of HER2-targeting therapy in homogeneously amplified lung cancers appear to be justified.Modern Pathology advance online publication, 17 August 2012; doi:10.1038/modpathol.2012.125.
[Show abstract][Hide abstract] ABSTRACT: Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumor. Translocation t(11;19)(q21;p13) involving the MECT1 and MAML2 genes has been suggested as a diagnostic marker in these tumors. To determine the specificity of 11q21 locus rearrangements for MEC, fluorescence in situ hybridization analysis with specific MEC-I Dual Color Break Apart Probe was performed on a tissue microarray containing samples from almost 1200 salivary gland adenomas and carcinomas. Rearrangements of 11q21 were observed in 40% of 217 MECs. The frequency of rearrangements decreased with tumor grade and was found in 53% of G1, 43% of G2, and 31% of G3 tumors (P=0.015). There were no 11q21 rearrangements found in other salivary gland carcinomas including 142 adenoid cystic carcinomas, 104 acinic cell adenocarcinomas, 76 adenocarcinoma not otherwise specified, 38 epithelial-myoepithelial carcinomas, 15 polymorphous low-grade adenocarcinomas, 18 basal cell adenocarcinomas, 19 myoepithelial carcinomas, 12 papillary cystadenocarcinomas, 6 salivary duct carcinomas, and 10 oncocytic carcinomas. Furthermore, all analyzed salivary gland adenomas, including 39 cases of Warthin tumor and control samples, either from the salivary gland or from other organs were negative for 11q21 rearrangements. It is concluded that MECT1-MAML2 gene fusion is a highly specific genetic alteration in MEC with predominance in low-grade and intermediate-grade tumors.
No preview · Article · Jul 2012 · Diagnostic molecular pathology: the American journal of surgical pathology, part B
[Show abstract][Hide abstract] ABSTRACT: Oncogene amplification is a key step in cell transformation towards malignancy. Chromosomal aberrations involving the long arm of chromosome 11, including amplifications at 11q13 and 11q22, have been previously reported in cervical cancer. While the role of the CCND1 gene as the driver gene for 11q13 amplification is well established in different tumor types, the significance of the 11q22 amplicon is less clear. The 11q22 amplicon corresponds to several putative target genes including the apoptose inhibitor BIRC2, recently detected as amplified in cervical cancer cell lines. To better understand the distribution and frequency of 11q amplification sites in uterine cervical carcinomas, we analyzed BIRC2 and CCND1 copy number changes using fluorescence in situ hybridization in a tissue microarray containing 238 cervical cancers. High-level amplification of BIRC2 was found in 12.9 % of tumors. Amplification of BIRC2 in cervical carcinomas was homogeneous as shown in corresponding whole tissue sections of amplified tumors at the tissue microarray. BIRC2 amplification was significantly more frequent than CCND1 amplification (2.1 %) in our cohort (p < 0.01), and amplification of both genes were independent from each other. BIRC2 amplification was associated with younger-patient age (p < 0.05) and squamous cell differentiation (p = 0.025) of cervix carcinomas. However, BIRC2 copy number changes were not related to tumor stage, grading and nodal status of cervical cancers. In conclusion, BIRC2 is amplified in a subset of squamous cell carcinoma of the uterine cervix. Further studies are necessary to evaluate possible prognostic effects of BIRC2 copy number gains in cervical carcinomas.
No preview · Article · Jun 2012 · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin
[Show abstract][Hide abstract] ABSTRACT: Infection with human papillomavirus (HPV) is linked to oropharyngeal cancer. This analysis investigated possible associations between HPV status, smoking history and survival outcome in patients with neck metastasis and carcinoma of unknown primary (CUP).
Registries at the Universities of Hamburg and Kiel were searched for patients with CUP diagnosed from 2002 to 2011 who had formalin-fixed and paraffin-embedded metastatic lymph node samples available. All patients underwent routine diagnostic procedures to establish the primary site and received radiotherapy (60Gy using conventional fractionation) with or without concurrent cisplatin-based chemotherapy depending on disease extent. Genotyping was performed using polymerase chain reaction; p16([INK4a]) expression was assessed using immunohistochemistry.
Sixty-three patients were included; 23 (37%) had HPV DNA/p16+ samples and 40 (63%) were negative for either/both markers. A high proportion of patients had a history of tobacco smoking; significantly fewer patients with HPV+/p16+ samples were smokers than those who were negative for either/both markers (61% vs. 90%, respectively; p=0.0067). There were no statistically significant differences between overall or recurrence-free survival in HPV+/p16+ patients vs. those negative for either/both markers. Overall survival appeared to be superior in patients with <10 pack-years smoking history and HPV+/p16+ disease.
This study, the largest to date investigating HPV status in head and neck CUP, identified HPV and p16 overexpression in over one-third of patients. Tobacco smoking history appeared to affect survival in HPV+/p16+ patients. Smoking status should be considered as a prognostic factor in patients with CUP, along with HPV DNA status.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of anti-apoptotic Bcl-2 plays a role in prostate cancer progression, particularly in transformation to androgen-independent disease. Androgen-independent prostate cancers have been shown to harbor Bcl-2 gene copy number gains frequently suggesting that this genetic alteration might play a role in Bcl-2 overexpression. The relation of Bcl-2 overexpression and copy number gains or translocation of the BCL-2 gene in prostate cancer under hormone-naïve conditions is unknown.
Prostate cancers of 3,261 hormone-naïve patients undergoing radical prostatectomy were arrayed in a TMA with one tissue core (diameter 0.6 mm) per tumor. Bcl-2 immunohistochemistry, analyzed for Bcl-2 expression level (negative, low, and high), was correlated with clinical, histopathological and molecular (Ki67, p53) tumor features, and biochemical failure. Cancers with high-level Bcl-2 expression were evaluated for genetic aberrations by fluorescence in situ hybridization (FISH).
Bcl-2 expression was significantly up-regulated in tumors with aggressive phenotype as indicated by high Gleason score (P < 0.0001), advanced stage (P < 0.0001), and high proliferation index (P = 0.0114). The different Bcl-2 expression levels translated into significantly different survival curves showing better outcome for patients with lower Bcl-2 levels. The prognostic information obtained from the anti-apoptotic Bcl-2 was independent from the proliferation index (Ki67) of the cancer. FISH analysis detected no copy number gains or translocation of the Bcl-2 gene.
Bcl-2 overexpression in prostate cancers under hormone-naïve conditions is not associated with increased copy numbers of the gene. This suggests that these frequently detected genetic alterations in androgen-independent tumors occur late in prostate cancer progression.
[Show abstract][Hide abstract] ABSTRACT: Women with triple-negative breast cancer (TNBC) do not benefit from endocrine therapy or trastuzumab. Chemotherapy is the only systemic therapy currently available. To reduce the elevated risk of disease progression in these patients, better treatment options are needed, which are less toxic and more targeted to this patient population. We performed a comprehensive analysis of potential targetable genetic aberrations affecting the receptor tyrosine kinase/RAS/MAPK pathway, which are observed at higher frequencies in adenocarcinomas of other organs. Sixty-five individual TNBCs were studied by sequence analysis for HER2 (exon 18-23), EGFR (exon 18-21), KRAS (exon 2), and BRAF (exon 15) mutations. In addition, a tissue microarray was constructed to screen for EGFR gene copy gain and EML4-ALK fusion by FISH. Triple-negative status was confirmed by immunohistochemistry and FISH on tissue microarray sections. EGFR and CK5/6 immunohistochemical analyses were performed for identification of the basal-like phenotype. In addition, mutation analysis of TP53 (exon 5-8) was included. Sequence analysis revealed HER2 gene mutation in only one patient (heterozygous missense mutation in exon 19: p.L755S). No mutations were found in EGFR, KRAS, and BRAF. High polysomy of EGFR was detected in 5 of the 62 informative cases by FISH. True EGFR gene amplification accompanied by strong membranous EGFR protein expression was observed in only one case. No rearrangement of the ALK gene was detected. Basal-like phenotype was identified in 38 of the 65 TNBCs (58.5 %). TP53 gene mutation was found in 36/63 (57.1 %) tumors. We conclude that targetable genetic aberrations in the receptor tyrosine kinase/RAS/MAPK pathway occur rarely in TNBC.
No preview · Article · May 2012 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: Multiple different biologically and clinically relevant genes are often amplified in invasive breast cancer, including HER2, ESR1, CCND1, and MYC. So far, little is known about their role in tumor progression. To investigate their significance for tumor invasion, we compared pure ductal carcinoma in situ (DCIS) and DCIS associated with invasive cancer with regard to the amplification of these genes. Fluorescence in situ hybridization (FISH) was performed on a tissue microarray containing samples from 130 pure DCIS and 159 DCIS associated with invasive breast cancer. Of the latter patients, we analyzed the intraductal and invasive components separately. In addition, lymph node metastases of 23 patients with invasive carcinoma were included. Amplification rates of pure DCIS and DCIS associated with invasive cancer did not differ significantly (pure DCIS vs. DCIS associated with invasive cancer: HER2 22.7 vs. 24.2%, ESR1 19.0 vs. 24.1%, CCND1 10.0 vs. 14.8%, MYC 11.8 vs. 6.5%; P > 0.05). Furthermore, we observed a high concordance of the amplification status for all genes if in situ and invasive carcinoma of individual patients were compared. This applied also to the corresponding lymph node metastases. Our results indicate no significant differences between the gene amplification status of DCIS and invasive breast cancer concerning HER2, ESR1, CCND1, and MYC. Therefore, our data suggest an early role of all analyzed gene amplifications in breast cancer development but not in the initiation of invasive tumor growth.
Full-text · Article · Oct 2010 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: The HER2 gene is amplified and overexpressed in about 15%-20% of breast cancers. For every newly diagnosed breast cancer HER2 testing is a standard routine procedure. This article focuses on a number of issues raised in the context of current HER2 testing in breast cancer. It particularly points out issues arising in the recently published ASCO-CAP (American Society of Clinical Oncology/College of American Pathologists) guideline recommendations for clinical testing of HER2. Despite the significant correlation between HER2 status determination by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), standard considerations of laboratory testing, such as test accuracy, reproducibility and precision as well as current data, favor FISH methods over IHC assay methods for the determination of HER2 status. Biological and technical considerations of HER2 testing are also important in clinical practice. For example, HER2 gene amplification is directly linked to the protein expression level in breast cancer; however, the HER2 protein is not consistently analyzed on formalin fixed tissues due to variability in fixation methods/times and the impact of this fixation on HER2 protein antigenicity. FISH is significantly less dependent on tissue fixation artifacts. Hence, FISH is more reproducible between both central and peripheral laboratories than IHC and is more accurate for HER2 measurement, as well as being more strongly correlated with responsiveness to trastuzumab and lapatinib treatment. Until other methods are able to ensure similar test accuracy, reproducibility, precision and predictive value, FISH is recommended as the primary HER2 testing modality for women with breast cancer who are candidates for HER2-targeted therapies.
[Show abstract][Hide abstract] ABSTRACT: Core needle biopsies (CNBs) are widely used to determine human epidermal growth factor receptor 2 (HER2) status in breast cancer. Recent publications reported up to 20% false-positive results on CNBs if immunohistochemistry (IHC) is compared with fluorescent in situ hybridization (FISH). To clarify, if confirmation of IHC positivity by FISH is generally required, we analyzed the reliability of IHC positivity on CNBs versus surgical specimens in a multi-institutional study.
Five pathologic laboratories contributed to this study by performing IHC on 500 CNBs and the corresponding surgical specimens overall. If IHC revealed score 2+ or 3+, HER2 status was confirmed by FISH in a central laboratory. We compared evaluation according to US Food and Drug Administration-approved scoring criteria and recently published American Society of Clinical Oncology (ASCO)-College of American Pathologists (CAP) guidelines.
CNBs scored 3+ revealed five false-positive results if scoring followed the US Food and Drug Administration criteria (five of 40; 12.5%) and two false-positives in terms of the ASCO-CAP criteria (two of 33; 6.1%). IHC was false negative in one CNB only. By contrast, IHC on surgical specimens revealed five false-negative results, but only one false-positive result (one of 35; 2.9%) if scored following US Food and Drug Administration-approved criteria. With the aid of the ASCO-CAP criteria, false-positive IHC results were obtained in only one of the five participating institutions.
IHC 3+ scores on CNBs proved to be reliable in four of the five participating institutions if scoring followed the ASCO-CAP criteria. Therefore, accurate determination of HER2 status in breast cancer is possible on CNB using the common strategy to screen all cases by IHC and retest only 2+ scores by FISH. Prerequisites are quality assurance and the application of the new ASCO-CAP criteria.
Full-text · Article · May 2010 · Journal of Clinical Oncology
[Show abstract][Hide abstract] ABSTRACT: Myeloid sarcoma of the breast is a rare manifestation of acute myeloid leukaemia (AML). This report describes a patient who was diagnosed with AML FAB M2. Molecular analysis showed evidence of an NPM1 mutation (subtype A) and internal tandem duplications of the FLT3 gene (FLT3-ITD). Eight months after allogeneic stem cell transplantation, the patient developed a palpable mass in the left breast initially suspected as breast carcinoma. Core needle biopsy of the lesion resulted in diagnosis of myeloid sarcoma. Molecular analysis of formalin-fixed specimens of the breast tumour confirmed the known FLT3 and NPM1 gene mutations. Immunohistochemically, an aberrant cytoplasmic staining pattern for NPM1 and overexpression of FLT3 were demonstrated. The myeloid sarcoma showed complete transient resolution following treatment with the kinase inhibitor sorafenib. However, the patient developed bone marrow relapse and died in fatal cerebral haemorrhage 1 year after initial diagnosis of AML. In summary, combined molecular and immunohistochemical examination of NPM1 and FLT3 is helpful in the diagnosis of extramedullary manifestations of AML in core needle biopsies.
No preview · Article · Apr 2010 · Journal of clinical pathology
[Show abstract][Hide abstract] ABSTRACT: Recently, amplification of PPFIA1, encoding a member of the liprin family located about 600 kb telomeric to CCND1 on chromosome band 11q13, was described in squamous cell carcinoma of head and neck. Because 11q13 amplification is frequent in breast cancer, and PPFIA1 has been suggested to contribute to mammary gland development, we hypothesized that PPFIA1 might also be involved in the 11q13 amplicon in breast cancer and contribute to breast cancer development. A tissue microarray containing more than 2000 human breast cancers was analyzed for gene copy numbers of PPFIA1 and CCND1 by means of fluorescence in situ hybridization. PPFIA1 amplification was found in 248/1583 (15.4%) of breast cancers. Coamplification with CCND1 was found in all (248/248, 100%) PPFIA1-amplified cancers. CCND1 amplification without PPFIA1 coamplification was found in additional 117 (4.7%) tumors. Amplification of both PPFIA1 and CCND1 were significantly associated with high-grade phenotype (P = 0.0002) but were unrelated to tumor stage (P = 0.7066) or nodal stage (P = 0.5807). No difference in patient prognosis was found between 248 CCND1/PPFIA1 coamplified tumors and 117 tumors with CCND1 amplification alone (P = 0.6419). These data show that PPFIA1 amplification occurs frequently in breast cancer. The higher incidence of CCND1 amplification when compared with PPFIA1, the lack of prognostic relevance of coamplifications, and the fact that PPFIA1 amplification was found exclusively in CCND1-amplified cancers suggest that PPFIA1 gene copy number changes represent concurrent events of CCND1 amplification rather than specific biological incidents.
Full-text · Article · Jan 2010 · Genes Chromosomes and Cancer
[Show abstract][Hide abstract] ABSTRACT: Tissue microarrays (TMAs) have become a standard tool for tissue-based research during the last decade. In cancer research, depending on the available data attached to the arrayed tissue, three main types of arrays are commonly manufactured. Prevalence TMAs have no further data available and are suited to estimate the frequency of the occurrence of a particular alteration. Progression arrays include tissues of different stages of disease, and are instrumental to study the role of a marker protein for tumor initiation, progression, or metastatic growth. Prognosis TMAs contain tissues with patient follow-up data. These TMAs are the key to uncover the clinical impact of molecular markers. In combination with normal tissue arrays representing healthy tissues, prevalence, progression, and prognosis TMAs allow for a rapid and comprehensive analysis of molecular markers in human cancers. TMAs are also successfully used for many noncancer applications, such as Alzheimer's or inflammatory disease research.
No preview · Article · Jan 2010 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: New high-throughput screening technologies have led to the identification of hundreds of genes with a potential role in cancer or other diseases. One way to prioritize the leads obtained in such studies is to analyze a large number of tissues for candidate gene expression. The TMA methodology is now an established and frequently used tool for high-throughput tissue analysis. The recipient block technology is the "classical" method of TMA making. In this method, minute cylindrical tissue punches typically measuring 0.6 mm in diameter are removed from donor tissue blocks and are transferred into empty "recipient" paraffin blocks. Up to 1,000 different tissues can be analyzed in one TMA block. The equipment is affordable and easy to use in places where basic skills in histology are available.
No preview · Article · Jan 2010 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Immunohistochemistry (IHC) is the gold standard methodology for in-situ protein expression analysis in tissue samples. The combination of IHC and tissue microarray (TMA) technology allows for the simultaneous analysis of hundreds of tissue samples with an unprecedented degree of experimental standardization. The same immunostaining protocols used for conventional large sections can be used for TMAs, including antigen retrieval procedures for staining of routinely archived formalin-fixed tissue samples. The development of optimal IHC protocols is highly important for TMA studies because minor protocol variations often have a marked impact on the outcome of the staining. Preabsorption and isotype-specific control experiments should be included as the last step in protocol development to proof target protein-specific binding. Such controls are particularly important for new antibodies with unknown staining patterns.
No preview · Article · Jan 2010 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Number of intratumoral mast cells predicts survival in various cancers. The prognostic significance of such mast cells in surgically treated prostate cancer is unknown.
Mast cell densities were determined in prostate cancer samples of more than 2,300 hormone-naïve patients using a tissue microarray format in correlation with clinical follow-up data. Mast cells were visualized immunohistochemically (c-kit). All patients were homogeneously treated by radical prostatectomy at a single institution.
Mast cells were present in 95.9% of the tumor samples. Median mast cell number on the tissue spot was 9 (range: 0-90; median density: 31 mast cells/mm(2)). High mast cell densities were significantly associated with more favorable tumors having lower preoperative prostate-specific antigen (P = 0.0021), Gleason score (P < 0.0001) and tumor stage (P < 0.0001) than tumors with low mast cell densities. Prostate-specific antigen recurrence-free survival significantly (P = 0.0001) decreased with decline of mast cell density showing poorest outcome for patients without intratumoral mast cells. In multivariate analysis mast cell density narrowly missed to add independent prognostic information (P = 0.0815) for prostate-specific antigen recurrence.
High intratumoral mast cell density is associated with favorable tumor characteristics and good prognosis in prostate cancer. This finding is consistent with a role of mast cells in the immunological host-defense reaction on prostate cancer. Triggering mast cell activity might expand immunotherapeutic strategies in prostate cancer.
[Show abstract][Hide abstract] ABSTRACT: Targeted therapy based on the understanding of the molecular principles of malignant transformation and tumour heterogeneity has resulted in substantial progress in breast cancer therapy. The success of targeted therapy depends on the selection of patients. At present the hormone receptor and HER2-status are the generally accepted predictive markers of response to endocrine and anti-HER2 therapy, respectively. It is the role of pathology to assure standardization and quality control of the determination. However, simply knowing that the target exists is not optimal to tailor adjuvant therapy. Currently, newer technologies are aimed at identifying molecular signatures that characterize breast cancer patients who are most likely to respond to specific targeted therapies. So far, the available gene expression assays (RT-PCR or microarray-based) are not sufficiently validated to recommend routine use. The promotion of development and consolidation of tissue-based predictive assays is one of the primary future tasks of pathology.
[Show abstract][Hide abstract] ABSTRACT: Neutral endopeptidase (CD10), an ectopeptidase bound to the cell surface, is thought to be a potential prognostic marker for prostate cancer.
Prostate cancer patients (N = 3,261) treated by radical prostatectomy at a single institution were evaluated by using tissue microarray. Follow-up data were available for 2,385 patients. The cellular domain (membranous, membranous-cytoplasmatic, and cytoplasmatic only) of CD10 expression was analyzed immunohistochemically and correlated with various clinical and histopathologic features of the tumors.
CD10 expression was detected in 62.2% of cancer samples and occurred preferentially in higher Gleason pattern (P < 0.0001). CD10 expression positively correlated with adverse tumor features such as elevated preoperative prostate-specific antigen (PSA), higher Gleason score, and advanced stage (P < 0.0001 each). Survival analyses showed that PSA recurrence was significantly associated with the staining pattern of CD10 expression. Outcome significantly declined from negative over membranous, membranous-cytoplasmatic, to exclusively cytoplasmatic CD10 expression (P < 0.0001). In multivariate analysis, CD10 expression was an independent predictor for PSA failure (P = 0.0343).
CD10 expression is an unfavorable independent risk factor in prostate cancer. The subcellular location of CD10 protein is associated with specific clinical courses, suggesting an effect on different important biological properties of prostate cancer cells. The frequent expression of CD10 in prostate cancer and the strong association of CD10 with unfavorable tumor features may qualify this biomarker for targeted therapies.
Preview · Article · Jan 2009 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Amplification of the gene encoding estrogen receptor-alpha occurs in about 20% of breast cancers and is an important mechanism for estrogen receptor overexpression in this tumor type. In ovarian cancer, overexpression of estrogen receptor protein has been described in more than two thirds of cases. To study a potential role of estrogen receptor-alpha gene amplification for estrogen receptor overexpression in ovarian cancer, a tumor tissue microarray containing 428 ovarian cancers was analyzed by fluorescence in situ hybridization for estrogen receptor-alpha gene amplification and immunohistochemistry for estrogen receptor expression. The estrogen receptor-alpha gene status was successfully determined in 243 of 428 arrayed cancers. Estrogen receptor gene amplification was found in 5 of 243 (2%) of tumors. Amplification levels were usually low, with 4-8 estrogen receptor-alpha gene copies. However, one case had a high-level amplification, with more than 30 estrogen receptor-alpha gene copies. All five amplified tumors were estrogen receptor positive, with 3 of 5 tumors showing highest (Allred score, 7-8) estrogen receptor levels. The data demonstrate that estrogen receptor-alpha amplification occurs only rarely in ovarian cancer.
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) is a protein involved in the tumor progression of many cancer types and is an important therapeutic target. To determine its role in prostate cancer, we analyzed 2,497 prostate cancers on the DNA and protein level.
Tissue samples from each tumor were brought into a tissue microarray and analyzed by immunohistochemistry and fluorescence in situ hybridization. A subset of cancers was also sequenced for EGFR exon 18 to 21 mutations.
Detectable EGFR expression was found in 18% of cancers and was significantly associated with high grade, advanced stage, and high risk for prostate-specific antigen recurrence in univariate analysis (P < 0.0001, each). Fluorescence in situ hybridization analysis with a dual-labeling probe for centromere 7 and EGFR showed increased EGFR copy number in 3.3% of cases. EGFR copy number gains were mostly due to an overrepresentation of the entire chromosome and were associated with EGFR protein expression (P < 0.0001), high grade (P < 0.0001), and advanced stage (P = 0.0056). Only one cancer had a high-level amplification (>20 EGFR gene copies per cell). This amplification was heterogeneous, involving only approximately 30% of the cancer volume. EGFR mutations were not found in 35 of the cases analyzed.
Increased EGFR expression is often seen in prostate cancer and is associated with poor prognosis. The significant association of EGFR copy number gains with protein expression argues for the significant role of minimal gene copy number changes for protein expression. Although EGFR expression was not an independent prognostic variable, the potential utility of anti-EGFR medications might be worth further investigation in EGFR-expressing prostate cancer.
Preview · Article · Nov 2007 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Using an Affymetrix 10K SNP array to screen for gene copy number changes in breast cancer, we detected a single-gene amplification of the ESR1 gene, which encodes estrogen receptor alpha, at 6q25. A subsequent tissue microarray analysis of more than 2,000 clinical breast cancer samples showed ESR1 amplification in 20.6% of breast cancers. Ninety-nine percent of tumors with ESR1 amplification showed estrogen receptor protein overexpression, compared with 66.6% cancers without ESR1 amplification (P < 0.0001). In 175 women who had received adjuvant tamoxifen monotherapy, survival was significantly longer for women with cancer with ESR1 amplification than for women with estrogen receptor-expressing cancers without ESR1 amplification (P = 0.023). Notably, we also found ESR1 amplification in benign and precancerous breast diseases, suggesting that ESR1 amplification may be a common mechanism in proliferative breast disease and a very early genetic alteration in a large subset of breast cancers.
[Show abstract][Hide abstract] ABSTRACT: HER-2 is the target for antibody based treatment of breast cancer (Herceptin). In order to evaluate the potential role of such a treatment in esophageal cancers, HER-2 amplification and overexpression was investigated in primary and metastatic cancers of the esophagus. A tissue microarray was constructed from 255 primary esophageal cancers (110 adenocarcinomas and 145 squamous cell carcinomas), 89 nodal and 33 distant metastases. Slides were analyzed by immunohistochemistry (HercepTest; DAKO) and fluorescence in situ hybridization (FISH; PathVysion; Vysis-Abbott) for HER-2 amplification and overexpression. Amplification was seen in 16/110 (15%) adenocarcinomas and in 7/145 (5%) squamous cell carcinomas. There was a strong association between HER-2 amplification and overexpression, especially in adenocarcinomas (P<0.0001, log rank). There was a 100% concordance of the HER-2 results in primary tumor and corresponding metastases in 84 analyzed pairs. Amplification was typically high-level with more than 10-15 HER-2 copies per tumor cell. Amplification was unrelated to survival, grading, pT, pN, pM or UICC stage. We conclude that esophageal adenocarcinomas belong to those cancer types with relevant frequency high-level HER-2 gene amplification clinical trials or individual case studies investigating the response of metastatic HER-2-positive esophageal cancers to Herceptin((R)) should be undertaken. The strong concordance of the HER-2 status in primary and metastatic cancers argues for a possible response of metastases from patients with HER-2-positive primary tumors to Herceptin.
[Show abstract][Hide abstract] ABSTRACT: Ductal carcinoma in situ (DCIS) is a heterogeneous disease that progresses to invasive cancer in 30-50% of the patients. Its natural history is poorly defined so that we are unable to identify cases of DCIS that do not progress to invasive carcinoma during an individual's lifetime. However, pathologic features of DCIS are nowadays the basis for the estimation of the prognosis and planning of therapy. Exclusion of microinvasion, characterization of nuclear grade, architecture, size and distribution of the DCIS, presence or absence of comedonecrosis as well as the assessment of surgical margins are relevant factors for local treatment. The determination of steroid hormone receptor status is indicated in patients considering tamoxifen therapy after breast conservation. It is advisable to evaluate the features according to internationally accepted guidelines with proven prognostic relevance and reproducibility. Nevertheless, better prognostic factors are needed to adapt the management of this increasingly diagnosed disease to the individual patient.
[Show abstract][Hide abstract] ABSTRACT: Osteopontin (OPN) and CEACAM1 have diverse biological functions in the uterus and placenta throughout the estrous cycle and pregnancy and have been shown to interact with integrin beta3. OPN is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. Recently we showed the expression pattern of OPN in gestational trophoblastic tumors. CEACAM1 is an adhesion molecule of the carcinoembryonic antigen family that we have recently found to be expressed in endometrial cancer and that has been shown to be down-regulated in colorectal, prostate, and breast cancer. In this study, immunohistochemistry and immunofluorescence with specific antibodies were performed on a series of 20 normal endometrial samples, 17 endometrial hyperplasias, and 43 endometrial carcinomas (28 endometrioid, 10 serous, and 5 clear cell carcinomas) to investigate the expression pattern and cell-type specific localization of OPN and to correlate it with the expression of CEACAM1. In addition, Western blot was performed on normal human endometrium and endometrial neoplasia. Strong OPN expression with a consistent cytoplasmic localization in epithelial glandular cells was observed in the normal human endometrium in 80% of the samples of the proliferative and secretory phase (score 8-12). Similar results could be found in endometrial hyperplasias. Strong expression of OPN could be observed in 29 (67.4%) of the 43 analyzed endometrial carcinomas. Of the 43 analyzed tumors, 18 (41.8%) were in the high score (8-12) category with a strong OPN expression level; 11 of 43 (25.5%) showed a moderate score (4-7) category. In endometrioid carcinoma with increasing malignancy grade, increasing areas with low OPN expression level or complete loss of OPN expression could be observed. In contrast, serous tumors showed a strong OPN expression level. Similar results could be found in Western blot analysis. CEACAM1 showed similar results and could be found to be coexpressed with OPN in normal human endometrium and in endometrial neoplasia as we showed using immunofluorescence. In this study, the different expression patterns of OPN in endometrial tumors could additionally support the biological diversity of endometrioid and serous carcinomas together with other markers. We suggest that OPN might play a different role in the pathogenesis of endometrial cancer (possibly as a functional complex with CEACAM1) and could be relevant for invasive growth of such lesions.
No preview · Article · May 2006 · International Journal of Gynecological Pathology
[Show abstract][Hide abstract] ABSTRACT: Downregulation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), a cell adhesion molecule with tumor suppressing properties has been observed in a high percentage of carcinomas of the endometrium and other malignancies. The mechanisms for the dysregulation and the role of hormones and cytokines on the expression of CEACAM1 in endometrial carcinomas is unknown. We therefore studied the effect of estradiol, medroxyprogesterone acetate (MPA), RU486, gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 on the expression in the non-expressing endometrial tumor cell lines Hec1B and Skut1B, respectively. No induction of CEACAM1 expression was observed in Hec1B endometrial adenocarcinoma cells in response to hormones and cytokines whereas treatment with TPA and calcium ionophore A23187 resulted in the strong expression of endogenous CEACAM1 on the mRNA and protein levels. In contrast, no induction of CEACAM1 expression was observed in endometrial mixed mesenchymal Skut1B cells. Studies of other members of the CEACAM family revealed that the re-expression in Hec1B carcinoma cells is restricted to CEACAM1 suggesting a cell type-specific and cell type-independent mechanism of CEACAM1 activation via the protein kinase C (PKC) pathway. Induction of CEACAM1 expression was dependent on protein kinase C protein synthesis and luciferase reporter assays with CEACAM1 promoter constructs demonstrated that the re-expression of CEACAM1 is regulated at the transcriptional level. This is the first report demonstrating that activators of PKC are able to specifically induce the expression of CEACAM1 in human carcinoma cells and our findings may provide a basis for the therapeutic inhibition of tumor growth in malignancies in which CEACAM1 is downregulated.
[Show abstract][Hide abstract] ABSTRACT: HER-2 belongs to a family of four transmembrane receptor tyrosine kinases that mediate growth, differentiation and survival of cells. HER-2 overexpression and amplification occurs in approximately 15 to 25 % of breast cancers and is associated with aggressive tumour behaviour. Herceptin (trastuzumab), a humanized monoclonal antibody directed against the extracellular domain of the HER-2 receptor, has been shown to have clinical activity in HER-2-positive advanced breast cancer when administered alone or in combination with chemotherapy. It has been approved for HER-2-positive metastatic breast cancer by the United States Food and Drug Administration in 1998 and in the countries of the European Union in 2000. Recently, promising results of the four randomized international multicenter trials evaluating the therapeutic benefit of Herceptin in the adjuvant treatment of HER-2-positive primary breast cancer have been reported. Data of the first planned interim analysis of the studies showed significantly improved disease-free survival in patients assigned to one year of Herceptin compared to the control groups even after short term follow up. These results caused an immediate wave of demand for Herceptin in adjuvant therapy. Results of these studies are critically reviewed. Furthermore, the available preliminary results from studies using Herceptin in the primary (neoadjuvant) therapy of HER-2-positive breast cancer are addressed and possible implications for HER-2 testing are discussed.
No preview · Article · Feb 2006 · Verhandlungen der Deutschen Gesellschaft für Pathologie
[Show abstract][Hide abstract] ABSTRACT: The t(14;18)(q32;q21) involving the MALT1/MLT and IGH genes has been identified recently as a recurrent abnormality in mucosa-associated lymphoid tissue (MALT) lymphomas. The frequency of secondary chromosomal aberrations in MALT lymphomas harboring the t(14;18) is largely unknown. We therefore analyzed six t(14;18)-positive MALT lymphomas (five parotid, one conjunctiva) by interphase fluorescence in situ hybridization for aneuploidies of chromosomes 3, 7, 12, 18, and X, gains or disruption of the CMYC/8q24 and BCL6/3q27 genes, as well as deletions of the retinoblastoma and TP53 tumor suppressor genes. Except for one MALT lymphoma of the parotid with trisomy 3, neither aneuploidies nor deletions were detected in any of our cases.
No preview · Article · Feb 2006 · Cancer Genetics and Cytogenetics
[Show abstract][Hide abstract] ABSTRACT: Due to the low incidence of salivary duct carcinoma (SDC), only limited data in regard to the biologic behavior of this tumor and its immunohistochemical characteristics are available. The authors analyzed the clinical, molecular, and genetic profile of SDC and identified prognostic factors.
The follow-up of 50 patients with SDC was obtained and paraffin-embedded tumor samples were examined immunohistochemically. In all samples, the expression of Ki-67, HER-2, and the oncoproteins p16 and p53 was examined immunohistochemically, followed by a mutation analysis of p16 and p53. The survival rate was calculated by the Kaplan-Meier method and prognostic variables were analyzed with the log-rank test.
SDC predominantly effected male patients (66%) in their 7th decade of life. SDC mainly occurred in the parotid gland (78%; submandibular gland, 12%; minor salivary glands, 10%). Approximately two-thirds of the patients (33 of 50) presented with a T3/T4 tumor. In 28 patients (56%), cervical lymph node metastasis was present at the time of diagnosis. Local disease recurrence was observed in 48% of patients an average of 17.4 months after initial treatment. Distant disease metastasis developed in 48% of patients an average of 29 months after initial treatment. The average overall survival period was 56.2 months. In the current study, 20.6% of the probes with positive HER-2/neu expression were (+++) positive. p53 was expressed in 83.9% of the tumor samples. In 11.8% of the tumor samples, there was a lack of p16 expression.
Mutations of the p53 gene were more frequent in tumor samples with (++) and (+++) immunoreactivity and mainly affected exons 7 and 8. A mutation of the p16 gene was only found in 1 tumor sample. Expression of HER-2/neu and p53 was statistically linked (P < 0.05) to early local disease recurrence, distant disease metastasis, and survival rates.
[Show abstract][Hide abstract] ABSTRACT: New high-throughput screening technologies have led to the identification of hundreds of genes with potential roles in cancer or other diseases. For evaluation of promising candidate genes, however, in-situ analysis of high numbers of clinical tissue samples is mandatory. The tissue microarray (TMA) technology greatly facilitates such analysis. In this method minute tissue samples (0.6 mm in diameter) from up to 1000 different tissues can be analyzed on one microscope glass slide. All in-situ methods suitable for histological studies can be applied to TMAs without major changes in protocols, including immunohistochemistry, fluorescence in-situ hybridization, or RNA in-situ hybridization. Because all tissues are analyzed simultaneously with the same batch of reagents, TMA studies provide an unprecedented degree of standardization, speed, and cost efficiency.
Preview · Article · Feb 2005 · Methods in molecular medicine
[Show abstract][Hide abstract] ABSTRACT: Giant cell tumor (GCT) offers a unique model for the hematopoietic-stromal cell interaction in human bone marrow. Evidence has been presented that GCT stromal cells (GCTSCs) promote accumulation, size and activity of the giant cells. Although GCTSCs are considered the neoplastic component of GCT, little is known about their genetic basis and, to date, a tumor-specific gene expression pattern has not been characterized. Mesenchymal stem cells (MSCs) have been identified as the origin of the GCT neoplastic stromal cell. Using state of the art array technology, expression profiling was applied to enriched stromal cell populations from five different GCTs and two primary MSCs as controls. Of the 29 differentially expressed genes found, 25 showed an increased expression. Differential mRNA expression was verified by real-time polymerase chain reaction analysis of 10 selected genes, supporting the validity of cDNA arrays as a tool to identify tumor-related genes in GCTSCs. Increased expression of two oncogenes, JUN and NME2, was substantiated at the protein level, utilizing immunohistochemical evaluation of GCT sections and Western-blot analysis. Increased phosphorylation of JUN Ser-63 was also found.
No preview · Article · Jan 2005 · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin
[Show abstract][Hide abstract] ABSTRACT: The case of an 80-year-old woman who presented with epigastric symptoms is reported. Upper gastrointestinal endoscopy displayed Candida esophagitis and a localized swelling of the fundic mucosa. Histologic examination of the gastric biopsy showed a distinctive accumulation of numerous uniform plasma cells filled with so-called Russell bodies. On low-power view, the lesion resembled a neoplastic process due to the marked expansion of the lamina propria with distension of fundic glands. However, immunohistochemistry confirmed a polyclonal pattern of the plasma cells. This unusual reactive lesion of the gastric mucosa has only rarely been described and has been termed Russell body gastritis.
No preview · Article · Sep 2004 · Archives of pathology & laboratory medicine
[Show abstract][Hide abstract] ABSTRACT: Transplantation of retinal pigment epithelial (RPE) cells is discussed as a possible therapeutic approach for retinal degeneration. Xenogeneic transplantation of human RPE cells in animal models has been studied extensively. Various methods have been used to identify the graft cells, but these methods interfere with cell behaviour so that the monitored physiological post-transplantation course may be influenced. In the present study, we applied a method for an unequivocal identification of the graft cells without interfering cell metabolism or behaviour using in situ hybridisation (ISH) of human specific Alu sequences. Visualisation of the strong extended nuclear signal of Alu sequences was much easier than that of the small nuclear signals of donor-specific sex chromosome probes. With Alu probe, even single graft cells can be identified and their development can be observed in short-term and long-term studies. With this procedure, we could prove that donor cells were injected correctly into the subretinal space by a special injection technique that we developed previously. In combination with immunohistochemistry, donor cells could be clearly discriminated from macrophages, which contained phagocytosed donor cell fragments. Application of these ISH methods for species-specific identification was valuable for follow-up-studies of RPE transplantation.
No preview · Article · Feb 2004 · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin
[Show abstract][Hide abstract] ABSTRACT: Alterations of the retinoblastoma (RB1) tumor suppressor gene are not only associated with retinoblastoma but also with several other malignancies including osteosarcoma. Besides direct sequence alterations, hypermethylation of a CpG island in the promoter region can cause inactivation of the RB1 gene as it has been shown in retinoblastomas. We examined the methylation status of the RB1 gene in 25 osteosarcoma specimens by using the methylation-sensitive restriction enzymes SacII and SmaI. The restriction fragments were hybridized with clone p123, which is a 1.8-kb genomic subclone that spans the RB1 CpG island including the promoter region and exon 1. Whereas we reconfirmed hypermethylation of the RB1 gene in a sporadic retinoblastoma, no hypermethylation could be detected in the 25 osteosarcoma specimens, suggesting that hypermethylation of the RB1 promoter is not of major importance during osteosarcoma genesis.
No preview · Article · Jan 2004 · Pediatric Hematology and Oncology
[Show abstract][Hide abstract] ABSTRACT: Fibroblastlike stromal cells, which are always present as a component of giant cell tumor of bone (GCT), can be observed in both in vivo and cultured cell samples. Although they are assumed to trigger the cancer process in GCT, the histogenesis of GCT stromal cells is poorly understood. It is known that mesenchymal stem cells (MSCs) can develop to osteoblasts. Evidence has been presented that GCT stromal cells can also develop to osteoblasts. A connection between MSCs and GCT stromal cells was sought by using 2 different laboratory approaches. First, immunohistological analyses revealed that some of the same markers, detected by the SH2, SH3, and SH4 antibodies and the CD166 antigen, were found in GCT stromal cells as in the first developmental stages of osteoblast differentiation from the initial MSCs. These immunohistological findings could be confirmed by reverse transcriptase polymerase chain reaction. Second, cellular differentiation by morphology and lineage-specific staining offered evidence that not only osteoblasts, but also chondroblasts and adipocytes, could be cultured from stromal cells. The presented double approach indicates that GCT stromal cells can originate from MSCs.
[Show abstract][Hide abstract] ABSTRACT: Vasopeptidase inhibitors are a new class of compounds that inhibit both angiotensin-converting enzyme (ACE) and neutral endopeptidase. This study determined whether treatment with the vasopeptidase inhibitor omapatrilat (OMA) produced different effects on renal and cardiovascular structure compared with inhibition of ACE by enalapril (ENP) in rats with two-kidney, one clip hypertension (2K1C).
Hypertensive 2K1C rats were randomized into four groups and studied for another 8 weeks: no treatment, OMA, ENP or ENP combined with the diuretic hydrochlorothiazide (ENP + HCTZ). Albuminuria, vascular and renal histology as well as glomerular expression of transforming growth factor-beta (TGF-beta) were determined at the end of the experiment.
OMA decreased blood pressure slightly better than ENP. However, combination of ENP with a diuretic lowered blood pressure equally effective as OMA. OMA was numerically more efficient in reducing cardiovascular and renal hypertensive changes compared with ENP. In contrast, the combination of ENP + HCTZ was as efficient as OMA. However, OMA lowered overexpression of TGF-beta in the non-clipped kidney better than ENP or ENP +HCTZ. Antihypertensive therapy surprisingly decreased renal function as shown by increased plasma creatinine and urea and decreased creatinine clearance.
OMA is marginally more potent compared with ENP alone in lowering blood pressure and preventing cardiovascular and renal injury. This effect may be due to slightly better blood pressure reduction because addition of HCTZ enhances the cardio- and nephroprotective capacity of ENP. In contrast, OMA reduces TGF-beta overexpression in the non-clipped kidney better than ENP or ENP + HCTZ. Therefore, vasopeptidase inhibition is not superior to ACE inhibition in the prevention of cardiovascular and renal damage Goldblatt hypertension.
Preview · Article · Nov 2003 · Nephrology Dialysis Transplantation
[Show abstract][Hide abstract] ABSTRACT: The CO2-laser-surgical reduction of the hyperplastic nasal turbinates represents a standard procedure in ENT-Surgery. In the presented paper, the authors introduce a new technique for the treatment of hyperplastic turbinates.
The laser beam is transmitted through a 90 degrees handpiece to the mobile "infra-guide" wave guide, which contains a 1 mm ceramic tube. By application of mirrow pieces with different angles, laser surgical reduction of those parts of the nasal turbinates is possible, which normally can not be reached by conventional, tangentially to the mucosa directed laser beams. The direction of the laser beam is controlled either optically, as in endonasal sinus surgery, or monitored through a camera. A total of 36 patients (22 women, 14 men, mean age 32.7 years) with nasal turbinate hyperplasia and either impaired nasal breathing and/or retronasal drip were treated with the new method.
The laser wave guide represents a minimally invasive and basically complication-free method feasable on an outpatient basis. As a particular advantage, laser surgical reduction of the posterior parts of the inferior and also the middle turbinate is possible. The majority of the patients relates of an improvement of the preoperative symptoms (regarding reduction of impaired nasal breathing: 61.3 %, regarding reduction of post nasal drip 87.1 %).
The laser wave-guide represents a basically complication-free, minimally invasive supplement to the different treatment modalities of the hyperplastic nasal turbinates.
No preview · Article · May 2002 · Laryngo-Rhino-Otologie
[Show abstract][Hide abstract] ABSTRACT: In the differential diagnosis of mass lesions of the salivary glands, myoepithelial sialadenitis (MESA), i. e. benign lymphoepithelial lesion, carries particular importance because of its association with Sjoegren's syndrome and development of malignant lymphoma. In the present study, epidemiology and clinical findings were analysed in relation to presence of MESA, Sjoegren's syndrome and lymphoma development.
67 patients, histopathologically classified by the salivary gland registry, were analysed retrospectively in regard to their clinical presentation, especially in regard to the chronical process of inflammation as present in MESA.
MESA primarily affects women in the 5th and 6th decade and regularly the parotid gland; in 44.8 % of the cases, there is multiple organ presentation. Xerostomy (38.5 %) is usually present (in 88,9 % of all cases) before or at clinical onset of gland inflammation, whereas xerophthalmy (28.4 %) did not show such a correlation. In general, rheumatic diseases (23.9 %) precede the gland-symptoms in 77.8 % of the patients. In 31.3 % of the cases a Sjoegren's syndrome was present. 26.9 % of the patients developed a malignant Non Hodgkin's Lymphoma (88.9 % of the MALT-type).
The most important clinical relevance of MESA lies in the higher probability to develop malignant lymphoma; this requires adequate staging-procedures and proper histopathological examination of sialogenic and nodal masses, especially over the course of the disease.
No preview · Article · Mar 2002 · Laryngo-Rhino-Otologie
[Show abstract][Hide abstract] ABSTRACT: The salivary duct carcinoma (sdc) represents a rare variant of the group of adeno-carcinomas of the salivary glands. Histopathologically, it is marked by solid and cribriform cell nests with central necrosis, displaying distinct similarity with the ductal carcinoma of the breast, where prognosis can be correlated with Her-2 gene-amplification. Based on this histopathological similarity, the prognostic value of Her-2 gene amplification in SDC was examined in the presented pilot-study.
Four own patients with different clinical courses were examined in regard to their histopathological features, Her-2 gene-amplification and proliferation (Ki67).
Three of the four patients died tumor related 2.4, 5.5 and 8.2 years after initial diagnosis. The remaining patient died tumor-free 6 year after diagnosis (myocardial infarct). The two patients with an early recurrent disease and distant metastasis showed a high Her-2 expression and proliferation (Ki67), compared to the other two patients.
In the presented pilot-study a distinct correlation between Her2-gene-amplification, proliferation (Ki67) and clinical course could be observed. Additional analysis to evaluate this aspect seems rectified, especially under recognition of therapy decisions.
No preview · Article · Oct 2001 · Laryngo-Rhino-Otologie
[Show abstract][Hide abstract] ABSTRACT: In atopic eczema both in local inflammatory reactions and in peripheral blood high interleukin (IL) 4: interferon-gamma (IFN-gamma) production ratios have been demonstrated, indicating predominance of TH2 cell subsets resulting in increased IL-4 production and high serum IgE. The in vitro immunomodulatory effects of recombinant human soluble IL-4 receptor (rsIL-4R) on IL-4-stimulated lymphocyte proliferation, IgE and IFN-gamma production were studied in peripheral blood mononuclear cells from 10 patients with atopic eczema and seven healthy donors. In addition to control cultures (without any stimulus) and cultures with simultaneous application of rsIL-4R and IL-4, time-kinetic experiments were performed. We further investigated the influence of rsIL-4R on IL-4 production in staphylococcal enterotoxin B (SEB) stimulated peripheral blood mononuclear cells. Early addition of rsIL-4R to IL-4-stimulated peripheral blood mononuclear cells resulted in an increase in IFN-gamma production and in suppression of IL-4 induced proliferation and IgE secretion. Unexpectedly, rsIL-4R in combination with SEB exhibited an IL-4 protective effect with a significant increase in detectable IL-4 in the culture supernatants. The present data support the assumption that rsIL-4R might be a promising new immunomodulatory substance in the treatment of atopic eczema.
No preview · Article · Dec 1998 · British Journal of Dermatology
[Show abstract][Hide abstract] ABSTRACT: Glomerular accumulation of macrophages/monocytes (M/M) is a typical early feature in the course of anti-thymocyte serum (ATS)-induced nephritis. We have previously shown that glomerular synthesis and expression of monocyte-chemoattractant protein-1 (MCP-1) occurs before influx of M/M and a neutralizing anti-MCP-1 antibody reduced this cell infiltrate by one third. The present study was undertaken to test the effect of two angiotensin II type 1 (AT1) receptor antagonists, losartan and irbesartan, on ATS-stimulated MCP-1 expression as well as glomerular influx of M/M.
Treatment of rats with either losartan or irbesartan was started 24 h before administration of ATS. After 24 h, MCP-1 mRNA expression was evaluated by RT-PCR and Northern blots. MCP-1 protein was determined by Western blots and chemotactic factors released from isolated glomeruli were measured by chemotactic assay. Kidney sections were stained for rabbit IgG, complement C3, and M/M (ED1 antigen).
Both AT1-receptor antagonists caused a significant, but not total reduction in MCP-1 mRNA and protein expression 24 h after injection of ATS. Treatment with losartan or irbesartan also reduced the chemotactic activity of isolated glomeruli from nephritic animals. Quantification of ED1-positive cells revealed that losartan as well as irbesartan reduced glomerular M/M invagination in nephritic rats by approximately 30-50%. However, treatment with AT1-receptor antagonists did not influence binding of ATS to mesangial cells and subsequent complement activation indicating that the attenuated MCP-1 expression is not due to differences in delivery and binding of ATS to mesangial cells.
Our data indicate that short-term antagonism of AT1 receptors abolished the early glomerular MCP-1 expression and M/M influx. These results indicate that angiotensin II may exert immunomodulatory effects in vivo and adds a new mechanism showing how this vasopeptide may be involved in the pathogenesis of renal diseases.
No preview · Article · Mar 1998 · Experimental nephrology
[Show abstract][Hide abstract] ABSTRACT: The selection criteria for a nerve-sparing radical prostatectomy (NSRP) are not thoroughly investigated and are based mainly on preoperative digital rectal examinations and intraoperative findings. At our institution NSRP is performed only on patients whose preoperative systematic sextant biopsy of the prostate showed only unilateral cancer. To prove the safety of these criteria, we analyzed the incidence of positive surgical margins and tumor progression rate in patients who were selected for an NSRP only by the result of the biopsy.
Preoperative systematic sextant biopsies revealed unilateral cancer in 69 preoperatively potent men of 289 consecutive prostatic cancer patients (23.9%); contralateral NSRP was performed on these 69 patients. The prostate specimens were investigated by using a 3-mm step-section technique to identify positive surgical margins. Tumor progression was defined as a prostate-specific antigen (PSA) level greater than 0.4 ng/mL in the native and greater than 0.025 ng/mL in the suprasensitive postoperative blood test. Mean follow-up was 15 months (range 6 to 24).
In 69 patients who underwent NSRP, 11 positive margins (15.9%) were found. Only 3 patients (4.3%) had a positive margin on the nerve-sparing side. In 220 patients who underwent non-NSRP 59 positive margins (26.8%) were detected. PSA recurrence rate after 12 months was similar in patients with NSRP and non-NSRP. Analysis of systematic sextant biopsies gives safe selection criteria because in approximately 95% the surgical margin on the nerve-sparing side will be negative.
Basing the indication for an NSRP on the results of preoperative systematic biopsies was safe according to margin status and postoperative PSA, when all patients with tumor in one of the three biopsy cores of each side of the prostate were excluded from an NS technique on that side. Such a strict approach will exclude approximately 30% of patients from NSRP unnecessarily because of tumor findings on a prostate side where the cancer is still organ-confined. Less strict criteria, including patients with only well-differentiated cancer and a maximum of one positive biopsy on the evaluated side, seem to be as safe as the described selection. However, data on these patients need further evaluation.
[Show abstract][Hide abstract] ABSTRACT: Infarct size delineation by triphenyltetrazolium chloride (TTC) staining is dependent on sufficient reperfusion. We therefore evaluated the possibility of using propidium iodide (PI), a reagent conventionally used in flow cytometry to fluorescently stain dead cells, for infarct size analysis after short periods of reperfusion. Forty-five rabbits were subjected to either 15 min, 2 h or 4.5 h of coronary artery occlusion without reperfusion, or to 15 min, 30 min and 2 h of coronary artery occlusion followed by 30 min, 1 h and 3 h of reperfusion. Fifteen min before terminating the experiment, PI was injected into the left atrium. Patent blue violet was used to delineate the area at risk. Following incubation in TTC, the area at risk was excised and cross sections obtained for microscopical infarct size quantification by PI fluorescence. PI fluorescence was absent after permanent occlusion and in control areas. Infarct sizes measured by TTC staining were significantly smaller after 1 h of reperfusion as compared to 3 h of reperfusion (30 min occlusion: 1+/-1 v 34+/-9%; P<0.05; 2 h occlusion: 9+/-6 v 47+/-8%; P<0.01). In contrast, infarct sizes determined by PI fluorescence reached values comparable to those measured by TTC staining or conventional histology after longer times of reperfusion already after 30 min of reperfusion (30 min occlusion: 35+/-16.5%; 2 h of occlusion: 61+/-8%). Therefore, after short times of reperfusion infarct size measurement by PI fluorescence is more reliable than by TTC staining.
No preview · Article · Aug 1997 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: The creation of transmyocardial channels from the epicardium to the left ventricular cavity with the use of a CO2 laser is a modern approach in the treatment of patients with chronic ischemic cardiac disease. The histological features of human myocardium at different times after transmyocardial laser therapy have not been previously described. We had the opportunity to examine hearts from patients who died without clinical evidence of a persistent therapeutic effect at 3, 16, and 150 days after transmyocardial laser revascularization (TMR) respectively.
We grossly localized the laser-created channels in unfixed and formalin-fixed tissue. Three ventricular levels were defined for cutting the hearts into four segments. Then, transmural blocks were excised and cut crosswise and lengthwise for histological investigation through the use of established staining methods. On day 3, laser-induced channels were filled with abundant granulocytes and thrombocytes, fibrinous network, and detritus and were surrounded by severe myocardial necrosis. Furthermore, the epicardial and endocardial portions were obstructed by fibrinous network and microclots. Granulocytes were mostly absent on day 16; in addition, the channels were filled with erythrocytes or fibrinous network. On day 150, we observed a string of cicatricial tissue admixed with a polymorphous blood-filled capillary network and small veins, which very rarely had a continuous wrinkled link to the left ventricular cavity.
We found different stages of wound healing in human nonresponder myocardium after TMR, resulting in scarred tissue that displayed capillary network and dilated venules without evidence of patent and endothelialized laser-created channels. Experimental studies are necessary to analyze the morphological basis for TMR-mediated effects in human responder myocardium.
[Show abstract][Hide abstract] ABSTRACT: The complement system has been suggested to play a role in reperfusion injury which may result from an enhanced destruction of myocardial tissue or from an impairment of reflow. We investigated the influence of the C5b-9 complement complex on infarct size, reflow and arrhythmogenesis.
Twenty-eight C6-competent rabbits and 18 rabbits with congenital C6 deficiency were subjected to either 30 min or 2 h of coronary artery occlusion followed by reperfusion. C6 deficiency was confirmed by the complement titration test and immunohistology. The triphenyl tetrazolium chloride method was used to delineate infarct size. Reflow into infarcted areas was evaluated histologically after an in vivo injection of propidium iodide which served as an early fluorescence microscopic marker of damaged myocardium subjected to reflow. Continuous ECG monitoring allowed the recording of arrhythmias.
After 30 min of coronary artery occlusion infarct size was significantly smaller in C6-deficient rabbits (5.0 +/- 2% of the risk region) as compared to C6-competent rabbits (28.4 +/- 8.5%, P = 0.0371). The extent of reflow into damaged myocardium was nearly the same in both animal groups at this time (38 +/- 9 vs. 39 +/- 7% of the risk region). After 2 h of coronary artery occlusion, infarct size was not different between both animal groups, but the extent of reflow into damaged myocardium was significantly smaller in C6-competent rabbits than in C6-deficient rabbits (25 +/- 4 vs. 40 +/- 4%; P = 0.0185). Two of the 18 C6-deficient rabbits had ventricular arrhythmias (Lown II-IV), none of which was fatal. Eleven of the 28 C6-competent animals had major ventricular arrhythmias which were fatal in 6 rabbits.
These results suggest that the lytic C5b-9 complement complex leads to reperfusion injury in the early phase (30 min) of ischaemia, resulting in a larger infarct. After 2 h of ischaemia, complement activation enhances the no-reflow phenomenon but does not affect infarct size. Finally, the C6 status seems to influence the susceptibility to ventricular arrhythmias after coronary artery occlusion, independent of reperfusion.
No preview · Article · Sep 1996 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: The salivary gland registry of the University of Hamburg (1965-1992) comprises a total of 327 mucoepidermoid carcinomas (MEC). MEC occur between the age of 5 and 96 years, most frequently in the sixth and seventh decade of life. The average age lies below 50 years (48.9 years). There is a slight predisposition of females (56.3%). Nearly 60% of all MEC care localised in the major salivary glands. The parotid gland is the most frequent localisation (53.5%) followed by the palate (17.4%). MEC are less frequent in the submandibular and sublingual glands, such as the minor salivary glands of the oropharynx and the cavity of the mouth. 53.2% of all MEC's can be classified as highly differentiated, the remaining 46.8% as poorly differentiated. MEC's of the palate are predominantly highly differentiated (72.0%). Among the variants of MEC, cystic (25.4%), clear cell (11.0%) and oncocytic (0.6%) tumours can be observed. MEC's of the clear cell type tend to originate from minor salivary glands (69.5%). MEC of childhood and adolescence (8.6%) are preferably seen in the parotid gland (46.4%) and the palate (39.3%). Up to the 20th year of life, cystic tumours and tumours of the clear cell type are comparatively more frequent.
No preview · Article · Oct 1994 · Laryngo-Rhino-Otologie
[Show abstract][Hide abstract] ABSTRACT: The mucoepidermoid carcinomas of the salivary glands (MEC) show histologically a wide spectrum of differentiation from solid epidermoid tumours to cystic mucous-filled tumours. Epidermoid cells, mucous cells and intermediate cells are the main cellular components. Besides clear cell (11.0%) and oncocytic (0.6%) variants and "cystic" (25.4%) tumours can be observed. According to the per cent of mucous cells and cystic spaces of the total tumour mass different degrees of histomorphologic differentiation can be distinguished. Therefore highly differentiated MEC (53.2%) with more than 50% mucous cells and cystic spaces, respectively low-differentiated MEC (46.8%) showing less than 50% mucous cells and cystic spaces can be subclassified. In respect of the therapeutic proceeding it should be recognized that the histopathological subclassification of MEC of the salivary glands (7) is based on morphological criteria only. It may not be equalized with a grading of malignancy since even highly differentiated MEC can grow infiltratively, sometimes osseo-destructively, to recur locally and to metastasize predominantly into the regional lymph nodes. Consequently the histopathological subclassification of MEC does not allow certain prognosis in individual cases.
No preview · Article · Oct 1994 · Laryngo-Rhino-Otologie
[Show abstract][Hide abstract] ABSTRACT: The terminal, membrane-damaging complement complex C5b-9 accumulates in the infarcted myocardium. In experimental myocardial infarction, we investigated the time course of C5b-9 deposition and the influence of reperfusion. In a group of 17 rabbits (group 1), the circumflex coronary artery was occluded for different time periods ranging from 0.5 to 29 h without subsequent reperfusion. A second group of 23 rabbits (group 2) underwent coronary artery occlusion for periods ranging from 0.5 to 6 h followed by reperfusion. C5b-9 was determined in transmural myocardial biopsies by immunohistochemistry and by ELISA. In group 1, C5b-9 accumulation in the ischaemic myocardium was found only after 5 to 6 h of coronary artery occlusion. In group 2 (ischaemia and reperfusion), significant C5b-9 deposition was already observed after 30 min of myocardial ischaemia. We conclude that in the absence of reperfusion C5b-9 accumulation occurs as a late event when most of the jeopardized myocardium has probably already become necrotic. In the presence of reperfusion, however, the complement system is activated rapidly and this could play a role in the pathogenesis of reperfusion injury.
No preview · Article · Apr 1994 · European Heart Journal
[Show abstract][Hide abstract] ABSTRACT: We report on the case of a seven-year old boy who developed an enlarged swelling of the oropharynx. Clinical examination revealed a well demarcated cystic tumour. Histopathologically the diagnosis of an intramuscular haemangioma could be made because of the multitude of vascular spaces filled with erythrocytes lying between bundles of smooth muscles. Such a lesion coming from the posterior wall of the oropharynx is not described in literature. In differential diagnosis the angiosarcoma and the arteriovenous aneurysm must have been involved since there were multiple arteries and veins lying close together in couples. Moreover the adenotomia carried out one and a half year earlier prompted us to think about the possibility of a traumatic arteriovenous fistula.
No preview · Article · Dec 1992 · Laryngo-Rhino-Otologie
[Show abstract][Hide abstract] ABSTRACT: The authors report a case of proliferating squamous epithelium in the necrotic edge of multifocal oncocytic adenomatous hyperplasia of the parotid gland. A correct histological diagnosis is of great clinical relevance and difficulty, since similar findings can occur in mucoepidermoid carcinoma. The various cell types in the parotid gland and their roles in neoplasia are discussed.
[Show abstract][Hide abstract] ABSTRACT: Enzymes and tissue antigens were localized on plastic embedded undecalcified bones and teeth using Technovit 7200 VLC (Kulzer, Germany). This resin is hard enough for cutting and grinding procedures on rotating plates with diamond layers. The pores between the diamond grains are not obstructed with this resin. The procedure described here permits localization of antigens in the soft tissues adjacent to, or in the biological hard tissues themselves and in dental implants (ceramic or metallic) on the light microscopic level. The undecalcified bone is fixed and embedded in plastic and cut at 100-150 microns. The slices are ground automatically by a grinding machine to a thickness of 5-10 microns. After application of the substrates for alkaline and acid phosphatases and the required dyes, the distribution of these enzymes can be demonstrated. Tissue antigens also can be detected with slightly modified standard techniques of immunohistochemistry and lectin histochemistry using the peroxidase technique or fluorescence microscopy.
No preview · Article · Feb 1991 · Biotechnic and Histochemistry
[Show abstract][Hide abstract] ABSTRACT: In our opinion ameloblastic fibro-odontoma seems to be a true neoplasia. Complex composite odontomas and compound composite odontomas represent hamartomatous malformations. The ameloblastic fibro-odontoma seems not to differentiate into a complex composite odontoma. Since ameloblastic fibro-odontomas bear great resemblance to common odontomas, it is suggested that all odontomas be sent to a qualified oral pathologist for microscopic examination.
No preview · Article · Feb 1991 · Deutsche Zahnärztliche Zeitschrift
[Show abstract][Hide abstract] ABSTRACT: The results of 73 implantations performed from 1.7.1986-30.6.1988 are reported in a first follow-up study. 82% reached an air-bone gap of less than 30 dB, 55% of less than 20 dB. In comparison with other materials recently reported the functional results are a little less favourable due to the high percentage of revision surgery in our patients. Only few complications had to be noticed: no single extrusion was observed, but in one case a nearly complete resorption of the DOP was seen. Two DOPs, removed by revision surgery, were examined histologically. Beside resorptive processes, bony changes as a sign of biointegration into the middle ear were found. On account of our first results, DOP seems very suitable for the reconstruction of a destroyed ossicular chain, especially in cases where the autologous ossicles are not available.
No preview · Article · May 1989 · Laryngo-Rhino-Otologie