Biomedical subjects

Arndt Hartmann

Publications and source records attributed to Arndt Hartmann.

At least 37 records · Page 2Linked to original sources

PubMed2006

Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi.

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

Adolescent↗

PubMed2006

MIB-1 and MCM-2 immunohistochemical analysis does not aid in identification of serrated colorectal polyps with abnormal proliferation.

We investigated the staining characteristics of serrated polyps with abnormal proliferation (SPAP) using MIB-1 and MCM-2 to determine if they could provide assistance in delineating SPAPs from traditional hyperplastic polyps (HPs). Using published morphologic criteria we reviewed H&E slides of 107 polyps from 80 patients. Thirty-nine (36.4%) polyps met the criteria for SPAP Within a given region, polyps in the transverse colon had the largest percentage of SPAPs (50.0%) followed by the right colon (40.9%). The majority of SPAPs (82.1%) and HPs (72.1%) showed MIB-1 staining confined to the basal third of the crypts. The majority of SPAPs (59.0%) and HPs (52.9%) showed MCM-2 staining extending into the apical third of the crypts. We do not recommend MIB-1 or MCM-2 staining to differentiate SPAPs from conventional HPs, since staining characteristics are not significantly different between the 2 groups, and frequent variable crypt staining within a given polyp is difficult to interpret.

Biomarkers, Tumor↗

PubMed2005

Expression levels of the putative zinc transporter LIV-1 are associated with a better outcome of breast cancer patients.

We investigated the expression pattern of the breast cancer associated gene LIV-1 on mRNA and protein level in 111 human breast cancer patients by in situ hybridization as well as immunohistochemistry and focused on the unknown potential of LIV-1 expression levels as a prognostic marker. To our knowledge, this is the first study on endogenous LIV-1 protein expression. Results of our study indicate that LIV-1 mRNA and protein expression levels are only weakly correlated, suggesting posttranscriptional regulatory mechanisms. Furthermore, LIV-1 mRNA quantity in combination with a positive ER status seem to represent a better marker than the progesterone receptor status according to the prognostic significance for relapse free survival (RFS). A negative correlation of LIV-1 protein levels with tumor size, grade and stage reflects an association of LIV-1 protein expression with less aggressive tumors. High LIV-1 protein expression seems to be associated with a longer relapse free and overall survival in breast cancer patients with invasive ductal carcinoma. This association, however, seems to be dependent from other prognostic markers. Our data suggest that LIV-1 is a promising candidate for a novel marker for breast cancer patients with better outcome. Furthermore, our study presents a revised cDNA sequence of LIV-1 and demonstrates the localization of endogenous LIV-1 in the endoplasmic reticulum.

Base Sequence↗

PubMed2005

Improved clonality analysis of multifocal bladder tumors by combination of histopathologic organ mapping, loss of heterozygosity, fluorescence in situ hybridization, and p53 analyses.

The clonality status of multifocal bladder tumors is still controversially discussed with experimental evidence for both monoclonality and field cancerization. Methodologically, loss of heterozygosity (LOH) and genomic sequencing analyses are widely used in clonality analysis of malignant tumors. In the present study, we used LOH analysis and genomic sequencing in combination with fluorescence in situ hybridization (FISH) and extensive histopathologic whole-organ mapping to determine the clonal relationship of multifocal bladder cancer disease. Tissue sections (1 cm(2)) covering the entire urothelial lining were systematically dissected from 2 cystectomy specimens (cystectomy 1, no urothelial lesions, bladder infiltration by a leiomyosarcoma of the vaginal wall; cystectomy 2, multifocal pT3G3 tumors). The location of each sample was documented (bladder mapping). Urothelial cells were microdissected for LOH (chromosomes 9, 17p) and FISH analysis (CDKI2 (9p21), FACC (9q22), p53 (17p13.1), and centromeric probes for corresponding chromosome). Exons 5 to 9 of the p53 gene were sequenced in all tumor samples. No chromosomal alterations were detected in the cystectomy specimen without urothelial malignancies. The tumor-bearing bladder showed an increasing frequency of deletions with increasing malignancy of the investigated lesions. LOH analysis detected deletions only on chromosomes 9p and 17p. In contrast, FISH analysis revealed deletions of all investigated genes at chromosomes 9p, 9q, and 17p in all samples analyzed (preneoplastic and neoplastic tissue). An identical p53 mutation in codon 281 was found in all 7 analyzable tumor samples. Combination of molecular data with histopathologic bladder mapping suggested a monoclonal development of the multifocal lesions mostly via intraurothelial migration. Our data strengthen the results from recently published studies that patients with advanced urothelial carcinoma seem to have a monoclonal panurothelial disease in most cases. FISH showed a much higher sensitivity for detection of chromosomal losses than classical LOH analysis, especially in preneoplastic and small lesions. Combining 3 molecular approaches together with histopathologic organ mapping presents a valuable tool to determine the clonality status of multifocal bladder tumors.

Chromosomes, Human, Pair 17↗

PubMed2005

Molecular evidence for progression of nephrogenic metaplasia of the urinary bladder to clear cell adenocarcinoma.

Nephrogenic metaplasia or nephrogenic adenoma of the urinary tract may present a diagnostic challenge in surgical pathology practice. Previous case reports suggest the possibility of nephrogenic metaplasia progressing to clear cell adenocarcinoma, but a malignant potential of nephrogenic metaplasia is generally not acknowledged. A case of a 70-year-old female patient with multiple recurrences of nephrogenic metaplasia of the urinary bladder and subsequent development of clear cell adenocarcinoma is described. Immunohistochemical studies help to differentiate the 2 entities. Results of molecular studies, particularly comparative genomic hybridization analysis, suggest clonal evolution of nephrogenic metaplasia to clear cell adenocarcinoma in this case.

Adenocarcinoma, Clear Cell↗

PubMed2005

A metachronous, atypical, multifocal renal oncocytoma with a concomitant renal cell carcinoma of the contralateral side and bilateral multifocal oncocytomas: two case reports and review of literature.

We present one case of a metachronous, atypical, multifocal renal oncocytoma with a concomitant chromophobe renal cell carcinoma (RCC) of the contralateral side and one case of bilateral and multifocal oncocytomas. Oncocytomas are benign renal tumours that rarely appear bilateral or multifocal or with coexisting RCC. A common pathogenic denominator of oncoytomas and RCC is being discussed. The first case was a 63 years old patient presenting with a history of nephrectomy for a pT1 G1 pN0 R0 papillary RCC 4 years prior to presentation, showed two tumours of a singular kidney. Upon nephron-sparing surgery one typical and one atypical oncocytoma with an invasion of the perinephric fat were found. Comparative genomic hybridisation was performed. Both tumours revealed genetic alterations with loss of genetic material on chromosome 1p. The second case was a 62 years old patient presenting with multifocal and bilateral renal tumours of undeclared dignity upon imaging. During open exploration all tumours could be removed by nephron-sparing surgery and were identified as oncocytomas. Again comparative genomic hybridisation was performed. All 4 tumours revealed genetic alterations with loss of genetic material on chromosome 1p, one of the tumours an additional loss of chromosome 10.

Adenoma, Oxyphilic↗

PubMed2005

Gene expression profiling of progressive papillary noninvasive carcinomas of the urinary bladder.

PURPOSE: The aim of the present study was to define gene expression profiles of noninvasive and invasive bladder cancer, to identify potential therapeutic or screening targets in bladder cancer, and to define genetic changes relevant for tumor progression of recurrent papillary bladder cancer (pTa). EXPERIMENTAL DESIGN: Overall, 67 bladder neoplasms (46 pTa, 3 pTis, 10 pT1, and 8 pT2) and eight normal bladder specimens were investigated by a combination of laser microdissection and gene expression profiling. Eight of 16 patients with recurrent noninvasive papillary bladder tumors developed carcinoma in situ (pTis) or invasive bladder cancer (> or = pT1G2) in the course of time. RNA expression results of the putative progression marker cathepsin E (CTSE) were confirmed by immunohistochemistry using high-throughput tissue microarray analysis (n = 776). Univariate analysis of factors regarding overall survival, progression-free survival, and recurrence-free survival in patients with urothelial bladder cancer was done. RESULTS: Hierarchical cluster analyses revealed no differences between pTaG1 and pTaG2 tumors. However, distinct groups of invasive cancers with different gene expression profiles in papillary and solid tumors were found. Progression-associated gene profiles could be defined (e.g., FABP4 and CTSE) and were already present in the preceding noninvasive papillary tumors. CTSE expression (P = 0.003) and a high Ki-67 labeling index of at least 5% (P = 0.01) were the only factors that correlated significantly with progression-free survival of pTa tumors in our gene expression approach. CONCLUSIONS: Gene expression profiling revealed novel genes with potential clinical utility to select patients that are more likely to develop aggressive disease.

Aged↗

PubMed2005

Loss of p16 protein defines high-risk patients with gastrointestinal stromal tumors: a tissue microarray study.

Despite clearly defined histologic criteria, the prediction of tumor behavior for patients with gastrointestinal stromal tumors (GIST) still poses a challenge to pathologists. Therefore, searching for alternative markers that allow for better prognostic evaluation is an important task. To determine the practicability of immunohistochemical staining for p16 in clinical cases, we examined p16 protein expression in a group of 284 GISTs, a subset of which had long-term follow-up (median, 45 months; range, 1-204 months). P16 protein expression was ascertained on tissue microarrays as well as on standard sections. Survival analyses were carried out in 157 patients. P16 loss was found in 50% of GISTs, there being no correlation with age, sex, histologic subtype, signs of necrosis, or metastases. Patients having p16-negative tumors had a worse prognosis than those with p16-positive tumors (P = 0.012) with a 2.3-fold relative increased risk of dying of disease. P16 loss identified a subgroup of gastric tumors with a worse prognosis (P = 0.03). The multivariate configural frequency analysis identified two "antitypes," whose observed frequency was found to be significantly lower than the expected frequency [i.e., marker combinations: p16 positive, no metastases, and death of disease and p16 loss, metastases, and still alive]. The "type" whose observed frequency was significantly higher than the expected frequency consisted of the following marker pattern: p16 loss, necrosis, and death of disease (P < 0.001). In the multivariate Cox regression analysis, p16 loss, necrosis, and metastases each had independent prognostic value. P16 loss is a common molecular abnormality in GISTs and might be used in routine diagnosis to identify patients with high-risk tumors.

Adolescent↗

PubMed2005

Elevated nuclear maspin expression is associated with microsatellite instability and high tumour grade in colorectal cancer.

Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high-frequency microsatellite instability (MSI-H) and 159 microsatellite stable colorectal cancers (MSS/MSI-L) by immunohistochemistry (IHC) and partly by relative quantitative real-time RT-PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI-H tumours compared to both MSS/MSI-L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI-H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT-PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI-H tumours (p = 0.013), de-differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies.

Biomarkers, Tumor↗

PubMed2005

Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer.

The determination of HER2/neu status in breast carcinomas has become essential for the selection of breast cancer patients for Herceptin therapy. Herceptin treatment is used in patients with metastatic breast carcinoma with HER2/neu protein overexpression detected by immunohistochemistry (IHC) or gene amplification analysed by fluorescence in situ hybridization (FISH). A multiparametric fluorescent approach based on the simultaneous detection of HER2/neu gene amplification and protein expression was established to increase the accuracy, and to improve the reproducibility, of HER2/neu diagnostics. Based on four paraffin-embedded breast cancer cell lines, a combined fluorescent immunostaining (FIHC) and FISH method was developed by using the PathVysion HER2 DNA Probe Kit (VYSIS) and the polyclonal antibody from the HercepTest (DAKO). Diagnostic applicability was documented on 215 formalin-fixed primary breast carcinomas. Criteria for immunofluorescence quantification were chosen by analogy with the FDA-approved HercepTest scoring, ranging from 0 to 3+. There was 97.7% concordance between conventional IHC and fluorescence IHC. The FISH data resulting from the multiparametric approach did not differ from conventional FISH. Breast carcinomas with HER2/neu protein overexpression and simultaneous gene amplification were detected with 100% sensitivity. In addition, five of the 215 cases (2.3%) had HER2/neu gene amplification without protein overexpression. The main advantage of this novel approach is that polysomy, aneuploidy, gene amplification, and protein content can be analysed simultaneously in the same cell.

Adult↗

PubMed2005

Deletions of chromosomes 9 and 8p in histologically normal urothelium of patients with bladder cancer.

OBJECTIVES: Multifocality and frequent recurrence of urothelial carcinoma of the urinary bladder indicate a disease of the entire "urothelial field", but little is known about chromosomal alterations in histologically normal urothelium. Histopathologically normal urothelium from patients with and without concurrent urothelial carcinoma was analyzed for loss of heterozygosity (LOH) on chromosomes 8p, 9 and 17p, regions that are known to play an important role in urothelial carcinogenesis. MATERIALS AND METHODS: Histologically inconspicuous urothelial mucosa samples (n = 160) from cystectomy specimens of patients with urothelial carcinoma (n = 15, max. diagnosis: CIS, pT1-4; all tumors grade 3) were studied. Control samples (n = 50) were obtained from patients with benign prostatic hyperplasia (n = 30) and from cystectomies performed for invasive non-urothelial carcinoma (n = 20). Tissue samples were laser-microdissected and DNA was isolated using standard protocols. LOH analyses were performed using 16 polymorphic markers on chromosomes 8p, 9p/q and 17p. RESULTS: All investigated samples were informative for at least one microsatellite marker on each chromosome/chromosomal arm. Deletions were found on chromosome 9 and/or 8p in 15/160 (9.4%) of normal urothelial samples from urothelial cancer patients. These alterations were only found in 5/15 patients with urothelial carcinoma. There were no deletions on chromosome 17p. The marker D9S1113 on chromosome 9q33-34 was most frequently affected (11/15 samples, 73%). No chromosomal deletions were found in any of the 50 urothelial control samples. CONCLUSION: Specific genetic alterations frequently associated with bladder cancer are detectable in histologically normal urothelium of patients with bladder cancer, supporting the field effect hypothesis in urothelial carcinogenesis. However, intramucosal spread of tumor cells that escape light microscopic detection remains a possibility, since normal urothelium and concurrent carcinomas showed matching deletions. Chromosomal deletions in normal bladder mucosa are not a common finding and argue against a frequent genomic alteration of the entire urothelial field in bladder carcinogenesis.

Aged↗

PubMed2005

Microsatellite analysis of pleural supernatants could increase sensitivity of pleural fluid cytology.

Pleural effusions may result from various inflammatory, hemodynamic, or neoplastic conditions. A common diagnostic problem lies in distinguishing malignant from benign pleural effusions using routine cytological evaluation. We studied pleural fluid samples obtained from 14 patients with histologically confirmed malignancy and from 6 patients with benign pleural effusions using 12 microsatellite markers from 8 different chromosomal regions. Supernatants and cellular sediments of all 20 pleural fluid samples were analyzed. Routine cytological examination was 100% specific for malignancy but was only 57% sensitive. Microsatellite analyses of pleural fluid supernatants showed genetic alterations in tumor patients only. However, 50% of pleural effusions that were considered negative for malignancy by routine cytological analysis showed either loss of heterozygosity or microsatellite instability. The sensitivity of pleural fluid examination rose to 79% when routine cytological assessment was supplemented by molecular studies. Our data suggest that microsatellite analysis increases the sensitivity of cytological pleural fluid examination in assessing potential malignancy and that combining cytological and molecular methods may improve yield and certainty in diagnostically challenging cases.

Aged↗

PubMed2005

Patterns of p53 mutations separate ovarian serous borderline tumors and low- and high-grade carcinomas and provide support for a new model of ovarian carcinogenesis: a mutational analysis with immunohistochemical correlation.

The infrequent association of serous borderline tumors (SBTs) with invasive serous carcinoma has led to the view that SBTs are unrelated to invasive serous carcinoma. Nonetheless, mortality associated with SBTs is generally attributed to malignant transformation, and traditionally these tumors have been designated as "carcinomas of low malignant potential." Previous immunohistochemical studies evaluating p53 expression and molecular genetic studies evaluating mutational status have reported that p53 overexpression and mutations are infrequent in SBTs and occur in as many as 50% to 80% of invasive serous carcinomas. The different methodologies for determining p53 status and the failure to correlate the findings with tumor grade make these studies difficult to interpret. The current study was undertaken to overcome these deficiencies and to reconcile the relationship of SBTs to invasive serous carcinoma by performing a morphologic, immunohistochemical, and molecular genetic analysis comparing SBTs with low- and high-grade serous carcinoma. The molecular genetic analysis used a highly stringent, carefully designed nucleotide-sequencing method. A total of 96 sporadic serous tumors including 25 SBTs (11 atypical proliferative serous tumors and 14 intraepithelial low-grade serous carcinomas [noninvasive micropapillary serous carcinomas, MPSCs]), 12 low-grade serous carcinomas (invasive MPSCs), and 59 high-grade serous carcinomas were analyzed for their p53 mutational status of exons 5 to 9. Functional mutations, defined as mutations resulting in the alteration of the structure of the encoded protein, were detected in 30 of 59 (50.8%) high-grade serous carcinomas and 1 (8.3%) of 12 low-grade invasive serous carcinomas compared with 2 (8%) of 25 SBTs, both of these in intraepithelial low-grade serous carcinomas (noninvasive MPSCs). The similar frequency of p53 mutations in SBTs and low-grade invasive serous carcinomas in contrast to the significantly higher frequency of p53 mutations in high-grade serous carcinomas (P < 0.0005) suggests a common lineage for SBTs and low-grade invasive serous carcinomas and supports the view that SBTs are unrelated to the usual type of invasive serous carcinoma, which is a high-grade neoplasm. Mutational status was also correlated with p53 immunoreactivity. Although p53 immunoreactivity is generally higher in those specimens containing mutant p53, immunostaining is neither sufficiently specific nor sensitive enough to predict p53 mutations. The molecular genetic findings confirm our hypothesis of dual pathways of serous carcinogenesis based on previous analyses of KRAS and BRAF mutations on the same set of cases in which KRAS and BRAF mutations were found in 60% of SBTs and low-grade serous carcinoma but not in high-grade serous carcinomas. Based on these studies, we have proposed a model of serous carcinogenesis in which SBTs are the precursors of low-grade serous carcinomas whereas the usual type of invasive serous carcinoma is a high-grade neoplasm that develops "de novo" from in situ alterations in epithelial inclusion cysts.

Base Sequence↗

PubMed2005

Diagnosis of urothelial carcinoma of the bladder using fluorescence endoscopy.

Bladder cancer is a frequent disease and represents the second most common genitourinary neoplasm. Although many aspects of the management of superficial bladder cancer are now well established, significant challenges remain, which influences patient outcome. Early detection and treatment of recurrent disease is required to optimize bladder preservation, reduce patient morbidity, and increase quality of life and survival. Fluorescence endoscopy, often referred to as 'photodynamic diagnosis' (PDD), with intravesical application of photosensitizing agents, has been developed to enhance the early detection of bladder cancer. There is growing evidence that PDD using 5-aminolaevulinic acid (ALA), hexyl-ALA ester or hypericin enhances the detection of bladder cancer, particularly of high-grade flat lesions. Furthermore, transurethral resection of bladder tumour under fluorescence guidance reduces the risk of recurrent tumours. However, the impact on the progression of disease remains unclear and must be investigated in prospective randomized trials.

Aminolevulinic Acid↗

PubMed2005

In situ expression patterns of melanoma inhibitory activity 2 in healthy and diseased livers.

UNLABELLED: Recently we identified a novel gene of the MIA gene family, melanoma inhibitory activity 2 (MIA2) and found that MIA2 mRNA is selectively expressed in hepatocytes. Here, we analyzed the in situ expression of MIA2 protein and mRNA in healthy and diseased livers to get first insights into the function of MIA2. METHODS: We analyzed liver tissue of patients with chronic hepatitis C (HepC) infection and hepatocellular carcinoma (HCC) as well as a human multi-tissue array, primary human hepatocytes and the hepatoma cell-lines HepG2, Hep3B and PLC by immunohistochemical staining and quantitative RT-PCR. In addition to MIA2, the expression of alpha-smooth muscle actin (alpha-sma), a marker for activated hepatic stellate cells (HSCs)/myofibroblast, was analyzed. RESULTS: Hepatocytes were confirmed as the exclusive cellular source of MIA2 expression, with a granular, cytoplasmatic staining pattern without enhancement at the cell membrane. In contrast, only low MIA2 expression levels were detected in most HCC and hepatoma cell lines. Only in HCC that contained fibrous stroma or thick hyalinized bundles, adjacent atypical hepatocytes revealed strong staining. In accordance, MIA2 expression was also upregulated in non-tumorous livers of patients with HepC and correlated with the staging of fibrosis. Interestingly, both in HCC and liver tissues of patients with HepC we found a correlation of MIA2 and alpha-sma expression. DISCUSSION: We define for the first time in situ expression patterns of MIA2 in healthy and diseased livers. Our data raise the hypothesis that activation of HSCs/myofibroblasts has influence on MIA2 expression in vivo, consistent with our previous in vitro findings. Since the staining pattern and the protein structure highly suggests that MIA2 is a secreted protein, it may possibly serve as a marker of hepatic fibrosis.

Adult↗

PubMed2005

RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid-induced protoporphyrin IX in vitro.

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line-specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 microg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.

Aminolevulinic Acid↗

PubMed2005

Microdissection for detecting genetic aberrations in early and advanced human urinary bladder cancer.

Laser microdissection is an essential method for the investigation of the multistep carcinogenic process in the urinary bladder. Reliable detection of tumor-specific alterations which can be compromised by the presence of normal cells, requires microdissection of pure tumor cell populations (>80%) to detect loss of heterozygosity (LOH) by either fluorescence in situ hybridization (FISH) or sequence analysis. Multiple molecular methods need to be performed in the course of studying often-small lesions. This chapter describes in detail the use of laser microdissection, whole-genome amplification by improved primer extension preamplification (I-PEP)-polymerase chain reaction, and subsequent LOH, FISH, and sequencing analysis in the investigation of urothelial tumors and their precursor lesions. The combination of the described methods allows a wide spectrum of molecular investigations of tumor cells and helps to understand the fundamental alterations involved in urothelial carcinogenesis.

Chromosome Aberrations↗

PubMed2005

Laser microdissection for microsatellite analysis in colon and breast cancer.

Microsatellite analysis is a frequently used method for detection of chromosomal deletions by loss of heterozygosity studies and for detection of microsatellite instability. For reliable microsatellite analyses, a tumor cell content of at least 80% is required. Therefore, laser microdissection is an important prerequisite for those studies, allowing the contamination-free isolation of morphologically defined pure tumor cell populations. The combination of exact microdissection and subsequent whole-genome amplification by improved primer extension preamplification polymerase chain reaction (I-PEP-PCR) facilitates the analysis of multiple microsatellite loci in small tumor samples. This is especially important for the investigation of malignant tumors with low tumor cellularity. This chapter describes in detail the use of whole genome amplification by I-PEP PCR and microsatellite analysis in laser microdissected specimens of colon and breast cancer.

Breast Neoplasms↗