BAGE hypomethylation, a new epigenetic biomarker for colon cancer detection.

Early detection of colorectal cancer is a decisive step in the successful and complete cure of the disease. Epigenetic markers, in particular, those based on aberrant DNA methylation, can be used to diagnose cancer. B melanoma antigens (BAGE) are a family of genes and truncated genes located in the heterochromatic regions of several human chromosomes. Our previous work showed that BAGE loci (i.e., genes and truncated genes) were hypermethylated in normal tissues and hypomethylated in 98% of human cancers. In the present study, we analyzed DNA methylation of the BAGE loci in 54 colon cancers and in neighboring histopathologic normal tissue samples. Using a combined bisulfite restriction assay, we showed that BAGE loci were hypomethylated in 81% of carcinoma samples. Colon cancer could be diagnosed with 94% specificity, 83% sensitivity, and 89% accuracy. No correlation was found between DNA methylation of BAGE loci and age, gender of patients, nor with the tumor stage or site. Based on the hypothesis that during neoplastic transformation, hypomethylation occurs in juxtacentromeric CpG islands, we suggest that other genes located in the heterochromatic compartment should be tested. These new markers enrich the list of currently studied epigenetic alterations in colon cancer and could be associated with hypermethylation markers to develop reliable diagnostic tests.

Case report: primary carcinoid tumor of the testicle without metastases in combination with testicular atrophy and testosterone deficiency.

The first case of testicular carcinoid was represented as an element of a benign cystic teratoma by Simon et al. J Urol 1954; 72: 892-894. It is a rare disease accounting for less than 1% of all testicular neoplasms. We report a case of carcinoid of the testis without carcinoid syndrome and metastasis but with testosterone deficiency based on a bilateral testicular atrophy, which has not been previously reported.

Comparative transcriptional and functional profiling of clear cell and papillary renal cell carcinoma.

Renal cell carcinoma (RCC) is known to effectively prevent immune recognition. However, little is known about the mechanisms that underlie this phenomenon. Thus, the identification of immunogenic molecules associated with RCC and the elucidation of the corresponding signaling pathways are crucial to the development of effective treatments. We performed transcriptional and functional profiling with cDNA microarrays (1070 cDNA probes) on a total of 17 RCCs, 11 clear cell and 6 papillary, and on corresponding normal tissue. Samples were clustered based on their expression profiles. We found a total of 45 genes to be regulated equally by both tumor types compared to the normal tissue. A set of 13 differentially expressed genes was identified between the examined tumor subtypes. Functional analysis was performed for both gene sets and showed a significant enrichment of cell surface genes regulated in both tumor subtypes. Within these we found five surface marker genes to be upregulated (TNFRSF10B, CD70, TNFR1, PDGFRB, and BAFF) which are involved in immune responses via the regulation of lymphocytes and can also induce apoptosis. Their overexpression in both tumor subtypes suggests a possible involvement in the immune escape strategies of RCC. The combination of transcriptional and functional profiling revealed potential target molecules for novel therapy strategies that must be studied in more detail.

Analysis of activated EGFR signalling pathways and their relation to laminin-5 gamma2 chain expression in oral squamous cell carcinoma (OSCC).

Overexpression of epidermal growth factor receptor (EGFR) was shown for the majority of squamous cell carcinomas. The EGFR expression correlates to tumour size, stage and cytoplasmic accumulation of the laminin-5 gamma2 chain (Ln-5/gamma2), which is known as a marker of invading tumour cells. There is only limited knowledge if and how EGFR signalling pathways are important for invasion-associated processes and for the regulation of Ln-5/gamma2. Therefore the distribution of phosphorylated Erk1/2, p38 MAPK and Akt was immunohistochemically defined in oral squamous cell carcinoma (OSCC) of different histological grade and compared to histological criteria of invasion and cytoplasmic Ln-5/gamma2 deposition. With raising histological grade, there is a slight increase in nuclear pErk1/2-stained tumour cells (P=0.398) and a loss of nuclear (P=0.593) and increased cytoplasmic staining (P=0.144) of pAkt mainly in invading OSCC cells. Nuclear pp38 MAPK could only be sporadically detected in few cases. In case of pErk1/2 and pAkt, only a partial co-localisation could be revealed in cases with abundant kinases and Ln-5/gamma2. Among the investigated kinases, only pAkt shows a relation to histological grade and invasion in OSCC. pErk1/2, pp38 MAPK and pAkt do not represent a direct link between EGFR and Ln-5 synthesis. Therefore, enhanced Ln-5/gamma2 may be a secondary phenomenon of EGFR-induced tumour cell proliferation and dissemination.

Identification of CD70 as a diagnostic biomarker for clear cell renal cell carcinoma by gene expression profiling, real-time RT-PCR and immunohistochemistry.

The underlying molecular mechanisms of renal cell carcinoma (RCC) are poorly understood and more reliable markers for early diagnosis are needed. Hence, alternative strategies for biomarker discovery with appropriate validation technologies have to be performed. To elucidate genesis and progression of RCC we used high parallel chip based gene expression profiling comparing normal and tumour tissues. We compared corresponding control and tumour tissue samples from 10 patients with clear cell RCC. We isolated RNA from histologically well characterised tissue sections and performed reverse transcription, labelling and linear RNA amplification. Samples were hybridised on microarrays containing 642 human cDNAs. Of the 352 differentially expressed genes found, CD70 and FRA2 were selected for further evaluation by real-time RT-PCR. The analysis all showed a high potential to discriminate between normal and tumour tissue. Moreover, increased CD70 mRNA expression in tumour cells could be correlated to its expression at the protein level. Immunohistochemistry (IHC) showed very strong expression of CD70 in all tumour samples but no expression in adjacent normal kidney tissue. With our combined approach we were able to identify CD70 as a new marker for RCC, which may be useful in the future for improved immunohistochemical diagnosis.

Identification of tumor entities of renal cell carcinoma using interphase fluorescence in situ hybridization.

PURPOSE: We developed a rapid interphase fluorescence in situ hybridization (FISH) test to differentiate renal cell carcinoma (RCC) based on known genetic alterations and verified the suitability of this test for practical use. MATERIALS AND METHODS: We composed 2 FISH test sets using 6 centromere specific and 2 region specific DNA probes of human chromosomes. Test set 1 contained centromeric probes for chromosomes 1, 2, 6 and 9, as labeled by 4 fluorescence dyes. For test set 2 we chose 3p24pter and 3p13p14 regions, and centromeric probes of chromosomes 7 and 17. Interphase nuclei of tumor specimens were prepared from 50 mum frozen tissue sections and fixed on slides. The 2 sets were hybridized simultaneously side by side on the same slide. RESULTS: Seven clear cell carcinomas, 8 papillary carcinomas, 7 chromophobe RCCs and 3 oncocytomas were analyzed by interphase FISH. Results were compared with comparative genomic hybridization findings and pathological reports. Genetic alterations were detected in 22 of 25 analyzed tumors by FISH. FISH findings absolutely correlated with comparative genomic hybridization results. Of the analyzed carcinomas 22 could be identified correctly. In 3 tumors the histological subtypes were revised. CONCLUSIONS: The results of this study demonstrate that the performed test set allows the accurate identification of RCC in 1 hybridization step. Therefore, FISH represents an effective method for the rapid classification of RCC.

The accuracy of 250 fine needle biopsies of renal tumors.

PURPOSE: In some cases of uncertain lesions in the kidney it would be helpful to perform biopsies for preoperative histopathological evaluation. In this study we evaluated the accuracy of and the impact on tumor management of core biopsy for histopathological evaluation of small solid renal masses. MATERIALS AND METHODS: After radical or partial nephrectomy 250 renal tumor biopsies were performed in 50 patients. All biopsies were performed by 1 urologist after preparation of the kidney ex situ on back table visually guided. Formalin fixed paraffin embedded biopsies were evaluated by 1 pathologist. RESULTS: In 49 of 50 cases (98%) we could define the malignant behavior of the tumor when performing 1 central and 4 peripheral biopsies of each tumor. In 85.2% the grading was correctly defined. A benign lesion was revealed in 4 cases (8%, all oncocytoma). In renal tumors 4 cm or smaller in diameter the accuracy of 1 central and 1 peripheral biopsy each regarding definition of tumor origin, tumor grading and cell type/growth pattern was 96% and 95.5%, 84% and 84.4%, and 87.5% and 89.5%, respectively. In renal tumors more than 4 cm in diameter the accuracy was 100% and 98.1%, 85% and 94.3%, and 71.4% and 88.7%, respectively. CONCLUSIONS: Core biopsy of renal lesions is accurate enough for histopathological evaluation and determination of therapeutic procedure. Additionally, biopsy could be used for identifying benign renal lesion for observation.

CD70: a new tumor specific biomarker for renal cell carcinoma.

PURPOSE: To date there have been no specific tumor markers available for the differential diagnosis of renal cell carcinoma (RCC). In an earlier study we identified high RNA expression of CD70 in clear cell RCC. CD70 is a type II transmembrane protein belonging to the tumor necrosis factor family. It represents the ligand for CD27, a glycosylated transmembrane protein of the tumor necrosis factor receptor family. To our knowledge the function of CD70 in solid tumors is not known. In the current study we analyzed CD70 protein expression in different RCC subtypes. MATERIALS AND METHODS: A total of 68 tumor samples of different histopathological subtypes were investigated by immunochemistry, including 41 clear cell, 19 papillary and 5 chromophobe RCCs, and 3 oncocytomas as well as their normal tissue counterparts. Immunochemistry was performed on frozen tissue samples using monoclonal antibody against CD70. RESULTS: None of the normal kidney tissues showed CD70 expression. In contrast, all clear cell RCCs expressed CD70 at a high level. Positive immunostaining was observed in 1 papillary (5%) and in 1 chromophobe (20%) RCC. Five papillary tumor samples (26%) showed focal staining in less than 5% of cells. All other samples were negative for CD70. CONCLUSIONS: Our study identified CD70 as a new specific tumor marker for clear cell RCC. This new marker can be used for differential diagnosis in cases of uncertain histological classification. The function of this protein in tumorigenesis and its use as a diagnostic marker in serum and urine or as a therapeutic tool must be investigated in further studies.

Frequent DNA hypomethylation of human juxtacentromeric BAGE loci in cancer.

The BAGE (B melanoma antigens) sequence family contains 15 nearly identical sequences that are in the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. BAGE loci are expressed in male germ tissue and in a high percentage of cancers and cancer cell lines. We analyzed the DNA methylation state of the sequences in or near the promoters of the BAGE loci by a quantitative bisulfite and PCR-based assay (multiplex COBRA) using MboI and HphI in 18 somatic tissue samples, 4 testis and 4 sperm samples, and 48 tumors and tumor cell lines. In 94% of the control somatic tissue samples, DNA was highly methylated in the analyzed regions. In contrast, 98% of tumor DNA samples displayed hypomethylation. Also, DNA from testes and sperm was hypomethylated in at least one of the BAGE loci. BAGE transcripts were observed in only 47% of the analyzed tumor samples. Consequently, we propose BAGE hypomethylation as a new, highly informative epigenetic biomarker for the diagnosis of cancer, whose hypomethylation in cancer may be causally related to that of juxtacentromeric satellite DNA.

mRNA expression and protein distribution of the unspliced tenascin-C isoform in prostatic adenocarcinoma.

The inclusion or omission of the alternatively spliced region in the tenascin-C (Tn-C) mRNA gives rise to the large (Tn-C(L)) or small (Tn-C(S)) variant, respectively. Tn-C(L) is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling in cancer. Tn-C(L) mRNA expression and protein distribution have been studied in 44 prostatic adenocarcinomas using RNA/RNA in situ hybridization supplemented by reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemistry (clone BC-2). While the Tn-C(L) protein was demonstrated within tumour stroma, Tn-C(L) mRNA expression was mainly observed in carcinoma cells, regardless of the histological grade of the tumour. Carcinoma cells containing Tn-C(L) mRNA were particularly localized at the tumour invasion front. Tn-C(L) mRNA was also identified in benign prostatic hyperplasia, where it was present exclusively in the basal cell layer, and in prostatic intraepithelial neoplasia in which there was partial loss of positive basal cells and increasing positivity of luminal cells. Furthermore, newly formed tumour blood vessels and inflammatory and stromal cells take part in the expression of Tn-C(L) and are involved in the formation of a provisional tumour matrix. It is concluded that deposits of Tn-C(L) indicate rebuilding processes in non-neoplastic as well as in neoplastic prostatic tissues. In respect of the Tn-C(L) synthesis in budding prostatic carcinoma cells, the results demonstrate that tumour cells can directly produce the ECM components of carcinoma stroma, creating conditions that facilitate the process of invasion.

Photodynamic therapy of virus-associated epithelial tumours of the face in organ transplant recipients.

PURPOSE: The benefit for organ recipients is still counteracted by the side effects of immunosuppression. Among other effects, there is a 50-250 times increased risk of developing malignant skin tumours. Because these malignomas are known to develop particularly aggressivly, there is a special need for an efficient therapy. Here we demonstrate the treatment response to aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in these patients. METHODS: Five organ recipients with multiple tumours of the face were multifocally treated with ALA-PDT (32 tumours in all). After topical application of ALA using a thermogel, irradiation was done with a 635 nm diode laser (Ceralas 635, Biolitec, Jena, Germany). After intervals of 2 weeks, 4 weeks, and 12 weeks, therapeutic efficacy was assessed. RESULTS: There was complete remission in 24 tumours (75%). In six tumours (18.8%) a second or third PDT session was necessary for complete clinical remission. In two tumours (5.6%, invasive squamous cell carcinomas) the lesions were refractory to PDT. CONCLUSIONS: ALA-PDT is a valuable therapeutic alternative for the treatment of multifocal skin tumours in organ-transplanted patients. Furthermore, we see a growing role of ALA-PDT also for patients with frequently relapsing tumours of the skin with known genetically determined tumourigenesis (Gorlin-Goltz syndrome).

Genetic profile of bone metastases in renal cell carcinoma.

OBJECTIVE: The aim of this study was to define specific genetic alterations which are common in bone metastases in renal cancer patients. METHODS: Tumor DNA from 31 metastases and 13 related primary tumors was extracted from paraffin embedded tissue sections. DOP-PCR was performed to amplify the whole DNA. After labelling by PCR, CGH was performed according to standard protocols. RESULTS: The mean number of aberrations per metastasis was 6.3 (1-13). Losses of chromosomes 3p (76%), 6 (20%), 8p (20%), 9 (34%), 14q (27%) and 18 (20%) as well as gains of chromosomes 5 (45%), 8q (34%) and 17 (27%) were detected frequently. Thirteen related primary tumors were also investigated. In 7 cases, at least one identical alteration was found in both primary tumor and metastases. In these cases, the number of alterations was mostly higher in primary tumors than in metastases without statistical significance. However, in general, the frequency of alterations was higher in metastases. CONCLUSIONS: Bone metastases from renal cell carcinoma are characterized by typical genetic alterations. Changes leading to metastasizing happen early in tumor pathogenesis. However, further accumulation of genetic changes occurs in metastases leading to a more complex genetic pattern which might be necessary for progression to clinically relevant metastases.

Genetic characterization of lung metastases in renal cell carcinoma.

Prognosis of patients with renal cell carcinoma (RCC) is mainly determined by metastases. The understanding of the metastatic process will give the basis for a differential diagnosis leading to an individual prognosis and to new therapeutical strategies. In order to define specific genetic alterations which are common in renal cancer metastases of the lung, we performed comparative genomic hybridization (CGH) on metastases and in some cases on their related primary renal tumors. For CGH, DNA was isolated from 2 or 5 paraffin sections (5 micro m). Tumor and normal (control) DNAs were amplified by DOP-PCR and labeled with biotin-dUTP and digoxigenin-dUTP, respectively. Hybridization and detection were carried out according to standard protocols. In 33 out of 40 metastases, genetic alterations were detected, most frequently these were losses of chromosomes 3p (74%), 8p (31%), 9 or 9q (34%), 14 [26%, 18q (40%) and gains of chromosome 5/5q 34%], 7 (31%) and 12 (26%). Combination of loss of 8p and gain of 8q occurred frequently. The mean number of aberrations per tumor was 8.1 (1-11). The comparison of alterations in related primary and metastatic tumors showed identical alterations in 5 out of 8 cases. This study demonstrates, that lung metastases from renal cell carcinoma are characterized by an accumulation of specific genetic alterations which show a clonal relationship to the related primary tumors.

Genetic subtyping of renal cell carcinoma by comparative genomic hybridization.

The prognosis of renal cell carcinoma (RCC) varies dependent on histologic tumor subtypes. However, differentiation between RCC types may sometimes be difficult on histologic grounds alone. Furthermore, the prognostic value of histologic parameters for the individual prognosis is limited. Additional information on the molecular level seems necessary to obtain more certainty in diagnostic and prognostic evaluation. By investigating genetic alterations in different RCC subtypes, we sought to obtain a genotype-phenotype correlation. Eighty-two clear-cell, 53 papillary, 23 chromophobe RCCs and 26 renal oncocytomas were investigated. Comparative genomic hybridization (CGH) was performed on DNA from paraffin-embedded tissue samples. DNA was isolated from tumor areas by microdissection and amplified by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). CGH was performed according to standard protocols. We were able to detect specific alterations in each RCC subtype: clear cell RCC showed -3p, +5/5q, -8p, -9, -14, -18; papillary (chromophilic) RCC gains of chromosomes 7, 17, 16, 3, 12; chromophobe RCC loss of chromosomes 1, 2, 6, 10, 13, 17, 21; renal oncocytomas loss of chromosomes 1/1p and 14. Furthermore, for clear cell RCC, it was possible to define alterations which are associated with metastatic disease: loss of 9, 10, 14. Our results demonstrate that each RCC subtype is characterized by distinct genetic alterations. The definition of genetic alterations seems helpful for a tumor typing especially when morphology is equivocal. Therefore, genetic analyses represent a powerful diagnostic and prognostic tool for RCC.

Immunohistochemical demonstration of the gamma2 chain of laminin-5 in urinary bladder urothelial carcinoma. Impact for diagnosis and prognosis.

The heterotrimeric molecule laminin-5 (Ln-5) represents a main protein of the epithelial adhesions complex. It links the basement membrane (BM) with the hemidesmosomes of the basal urothelial cells. The study was aimed to evaluate invasion associated changes of the epithelial adhesion complex in urothelial carcinoma (UC) monitored by immunohistochemical demonstration of the Ln-5 gamma2 chain. For correlation to UC phenotype and patients outcome, a semiquantitative immunohistochemical analysis of 100 routinely processed paraffin embedded samples (non-invasive and invasive UC) using the antibody D4B5 specific for the Ln-5 gamma2 chain was performed. An increased risk of death is associated with an increased Ln-5 loss from BM (P=0.001), an increase of stroma deposition (P=0.001), as well as an increase of cellular retention of Ln-5 protein (P=0.001) (Kruskal-Wallis test). As shown in multivariate analysis, in addition to tumor stage the cellular retention of Ln-5 is the most important prognostic parameter. In consequence, the modulation of Ln-5 is recommended as a diagnostic marker of invasive UC phenotype.

An improved version of the DNA Methylation database (MethDB).

Cytosine methylation is a characteristic property of prokaryotic and eukaryotic genomes. In the latter, it is indispensable for a healthy development of the organism and uncontrolled changes in the distribution of 5-methylcytosine (5mC) have been linked to severe disorders, in particular cancer. The growing scientific interest in DNA methylation has led to a considerable amount of data about this epigenetic phenomenon. In order to make these data readily available, we have established a dedicated database. The DNA Methylation database (MethDB) is currently the only public database for DNA methylation (http://www.methdb.net). This constantly growing database has become a key resource in the field of DNA methylation research. The database contains currently methylation patterns, profiles and total methylation content data for 46 species, 160 tissues and 72 phenotypes coming from a total of 6667 experiments (as of September 4, 2002). About 14% of the data have not been published elsewhere. These data can be conveniently searched and represented in different ways. Recently, we have included an on-line submission tool that permits the scientific public to directly enter new data into MethDB.

Clonal origin of multifocal renal cell carcinoma as determined by microsatellite analysis.

PURPOSE: The reported incidence of satellite tumor lesions in renal cell carcinoma (7% to 25%) suggests that there is a risk of local recurrence after nephron sparing surgery. It remains largely unknown whether small satellite tumors show malignant features and whether they are metastases from the primary tumor. Therefore, we determined the clonality of multifocal tumors by molecular genetic analysis. MATERIALS AND METHODS: A total of 19 multifocal clear cell renal cell carcinomas were investigated by microsatellite analysis using 6 markers for chromosome 3p, namely D3S1560, D3S1289, D3S1766, D3S1300, D3S1566 and D3S1663. Polymerase chain reaction was performed according to standard protocols, followed by gel electrophoresis and automated analysis using an automated DNA sequencer (Li-Cor, Lincoln, Nebraska). RESULTS: All primary clear cell tumors were characterized by loss of heterozygosity on 3p. Multifocal tumors showed identical microsatellite alterations with at least 1 marker in 17 of the 19 cases. In 2 cases different microsatellite patterns were detected in tumors from the same kidney. CONCLUSIONS: Identical loss of heterozygosity and shift patterns detected in different tumors in the same kidney strongly suggest that multifocal clear cell renal cell carcinomas have a common clonal origin in most cases. These findings indicate that satellite tumors are the result of intrarenal metastasis from the primary tumor. The clinical implications of these results must be correlated with the clinical disease course in patients with multifocal renal cell carcinoma.