Udo Schumacher

ERG Status Is Unrelated to PSA Recurrence in Radically Operated Prostate Cancer in the Absence of Antihormonal Therapy

Purpose: About 50% of prostate cancers have TMPRSS2–ERG fusions with concurrent ERG overexpression. The aim of this study was to determine whether clinical differences exist between ERG-positive and ERG-negative cancers in surgically treated patients not exposed to antihormonal therapy. A secondary aim was to search for differences between these tumor classes. Experimental Design: A tissue microarray containing samples from more than 2,800 prostate cancers with clinical data was analyzed for ERG alterations by immunohistochemistry and FISH. Results were compared with tumor phenotype, biochemical recurrence, and molecular features considered important for prostate cancer. The effect of ERG on androgen receptor (AR)-dependent transcription was analyzed in cell lines. Results: ERG expression was found in 52.4% of 2,805 cancers with a 95% concordance between ERG expression and ERG gene rearrangement detected by FISH. ERG expression was unrelated to clinical outcome and tumor phenotype. Differences in AMACR, Annexin A3, Bcl2, CD10, ALCAM, chromogranin A, epidermal growth factor receptor, HER2, mTOR, p53, and synaptophysin status were significant but minimal in absolute numbers. The most striking difference was found for AR expression, which was markedly higher in ERG-positive cancers. In vitro studies showed ERG-dependent impairment of AR-mediated transcriptional activity. Conclusions: The striking similarities between these two types of prostate cancers rules out a major impact of ERG on tumor aggressiveness in early, not hormonally treated cancer. The marked difference in AR levels between ERG-positive and -negative cancers supports a systematic difference in potential response to hormonal therapy as previously observed in clinical trials. Clin Cancer Res; 17(18); 5878–88. ©2011 AACR.

CEACAM1 Expression in Cutaneous Malignant Melanoma Predicts the Development of Metastatic Disease

PURPOSE The cell adhesion molecule CEACAM1 is involved in intercellular adhesion and subsequent signal transduction events in a number of epithelia. CEACAM1 downregulation has been demonstrated in colorectal and prostate carcinomas. This study sought to analyze whether its expression in malignant melanoma is associated with metastasis. PATIENTS AND METHODS CEACAM1 expression was immunohistochemically evaluated in 100 primary cutaneous malignant melanomas and correlated with metastasis in a 10-year follow-up. Furthermore, CEACAM1 expression was analyzed in metastatic lesions (11 distant metastases and six sentinel lymph node metastases). Univariate Kaplan-Meier analysis and multivariate Cox proportional hazard regression analysis adjusted for standard prognostic indicators were performed to assess the prognostic relevance of CEACAM1 expression. RESULTS A total of 28 of 40 patients with CEACAM1-positive primary melanomas developed metastatic disease, compared with only six of 60 patients with CEACAM1-negative melanomas. Often, the strongest CEACAM1 expression was observed at the invading front. In addition, CEACAM1 expression was preserved in the metastatic lesions. Kaplan-Meier analysis revealed a highly significant association between CEACAM1 expression and metastasis (P <.0001); multivariate Cox regression analysis, including CEACAM1 expression status adjusted for tumor thickness, presence of ulceration, and mitotic rate, confirmed that CEACAM1 is an independent factor for the risk of metastasis and demonstrated that the predictive value of CEACAM1 expression is superior to that of tumor thickness. CONCLUSION Expression of the cell adhesion molecule CEACAM1 in the primary tumors in melanoma patients is associated with the subsequent development of metastatic disease. This raises the possibility of a functional role for this cell adhesion molecule in the metastatic spread it indicates.

Overexpression of the cell adhesion molecule L1 is associated with metastasis in cutaneous malignant melanoma

Modulation of cell adhesion molecule expression plays a key role in melanoma metastasis. In particular, the expression of the cell adhesion molecule L1 has been associated with the metastatic phenotype in a murine model of malignant melanoma. However, no such association between L1 expression and metastasis has been investigated in a clinical study. Therefore, L1 expression was determined immunohistochemically in 100 cases of malignant melanoma and correlated with metastasis in a 10-year retrospective study. Furthermore, nine distant metastases and five sentinel lymph node metastases were analysed for their L1 expression. Additionally, the expression of alpha2,3 sialic acid residues, which are recognised by the siglec domain of L1, was determined by Maackia amurensis agglutinin (MAA) lectin histochemistry. The log-rank test between Kaplan-Meier curves revealed a positive association between L1 expression and metastasis (P<0.0001) and multivariate Cox regression analysis adjusted for tumour thickness, ulceration and mitotic rate confirmed the prognostic power of L1 in malignant melanoma. As alpha2,3 sialic acid residues were absent in melanoma cells, homotypic adhesion between melanoma cells via their siglec domain can be excluded, suggesting a different adhesive function of L1 during melanoma metastasis. The functional role of L1 was further stressed by the fact that its expression was preserved in metastatic lesions.

Establishment and Characterization of a Cell Line from Human Circulating Colon Cancer Cells

Circulating tumor cells (CTC) in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors including colon cancer. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in patients with cancer. However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of patients with colorectal cancer. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. Here, we describe, for the first time, the establishment of cell cultures and a permanent cell line from CTCs of one patient with colon cancer. The cell line designated CTC-MCC-41 has been cultured for more than one year, and the cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the patient with colon cancer and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem cell-like properties, and an osteomimetic signature, indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. The establishment of this first colon cancer CTC line allows now a wealth of functional studies on the biology of CTCs as well as in vitro and in vivo drug testing.

Expression of CEACAM1 in Adenocarcinoma of the Lung: A Factor of Independent Prognostic Significance

PURPOSE To evaluate the prognostic relevance of CEACAM1 and sialyl Lewis X expression in adenocarcinomas of the lung. PATIENTS AND METHODS Paraffin wax sections of 93 patients with adenocarcinomas of the lung who underwent surgery between 1990 and 1995 were immunohistochemically investigated using monoclonal anti-CEACAM1 and sialyl Lewis X antibodies. The clinical course of all patients was followed up for a minimum of 5 years. RESULTS Sixty-one tumors were classified as CEACAM1-positive, and 32 were classified as CEACAM1-negative. Patients with CEACAM1-positive tumors had a significantly poorer overall (P =.00025) and relapse-free (P =.00029) survival than those with CEACAM1-negative tumors. Only three patients did not express the sialyl Lewis X glycotope, whereas 90 tumors (97%) were sialyl Lewis X-positive. In multivariate Cox regression analysis, next to tumor stage and sex, only the expression of CEACAM1 was a significant independent prognostic factor for survival. CONCLUSION Expression of CEACAM1 was an independent prognostic factor in our patient population and can be used to stratify patients with adenocarcinomas of the lung into low-risk and high-risk groups. In contrast, the expression of sialyl Lewis X was of no prognostic relevance because it was expressed in 97% of all investigated tumors, and most likely has no influence on the function of CEACAM1 in this tumor entity.

Myeloperoxidase attracts neutrophils by physical forces

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocytes surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPOs affinity to both the endothelial and the leukocytes surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Morphometric analysis of intestinal mucins under different dietary conditions and gut flora in rats.

Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.

PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent.

The overexpression of the chemokine receptor CXCR4 plays an important role in oncology, since together with its endogenous ligand, the stromal cell–derived factor (SDF1-α), CXCR4 is involved in tumor development, growth, and organ-specific metastasis. As part of our ongoing efforts to develop highly specific CXCR4-targeted imaging probes and with the aim to assess the suitability of this ligand for first proof-of-concept studies in humans, we further evaluated the new 68Ga-labeled high-affinity cyclic CXCR4 ligand, 68Ga-CPCR4-2 (cyclo(D-Tyr1-[NMe]-D-Orn2-[4-(aminomethyl) benzoic acid,68Ga-DOTA]-Arg3-2-Nal4-Gly5)). Methods: Additional biodistribution and competitions studies in vivo, dynamic PET studies, and investigations on the metabolic stability and plasma protein binding were performed in nude mice bearing metastasizing OH1 human small cell lung cancer xenografts. CXCR4 expression on OH1 tumor sections was determined by immunohistochemical staining. Results: natGa-CPCR4-2 exhibits high CXCR4 affinity with a half maximum inhibitory concentration of 4.99 ± 0.72 nM. 68Ga-CPCR4-2 showed high in vivo stability and high and specific tumor accumulation, which was reduced by approximately 80% in competition studies with AMD3100. High CXCR4 expression in tumors was confirmed by immunohistochemical staining. 68Ga-CPCR4-2 showed low uptake in nontumor tissue and particularly low kidney accumulation despite predominant renal excretion, leading to high-contrast delineation of tumors in small-animal PET studies. Conclusion: The small and optimized cyclic peptide CPCR4-2 labeled with 68Ga is a suitable tracer for targeting and imaging of human CXCR4 receptor expression in vivo. The high affinity for CXCR4, its in vivo stability, and the excellent pharmacokinetics recommend the further evaluation of 68Ga-CPCR4-2 in a proof-of-concept study in humans.

Genomic deletion of MAP3K7 at 6q12-22 is associated with early PSA recurrence in prostate cancer and absence of TMPRSS2: ERG fusions

6q12-22 is the second most commonly deleted genomic region in prostate cancer. Mapping studies have described a minimally deleted area at 6q15, containing MAP3K7/TAK1, which was recently shown to have tumor suppressive properties. To determine prevalence and clinical significance of MAP3K7 alterations in prostate cancer, a tissue microarray containing 4699 prostate cancer samples was analyzed by fluorescence in situ hybridization. Heterozygous MAP3K7 deletions were found in 18.48% of 2289 interpretable prostate cancers. MAP3K7 deletions were significantly associated with advanced tumor stage (P<0.0001), high Gleason grade (P<0.0001), lymph node metastasis (P<0.0108) and early biochemical recurrence (P<0.0001). MAP3K7 alterations were typically limited to the loss of one allele as homozygous deletions were virtually absent and sequencing analyses revealed no evidence for MAP3K7 mutations in 15 deleted and in 14 non-deleted cancers. There was a striking inverse association of MAP3K7 deletions and TMPRSS2:ERG fusion status with 26.7% 6q deletions in 1125 ERG-negative and 11.1% 6q deletions in 1198 ERG-positive cancers (P<0.0001). However, the strong prognostic role of 6q deletions was retained in both ERG-positive and ERG-negative cancers (P<0.0001 each). In summary, our study identifies MAP3K7 deletion as a prominent feature in ERG-negative prostate cancer with strong association to tumor aggressiveness. MAP3K7 alterations are typically limited to one allele of the gene. Together with the demonstrated tumor suppressive function in cell line experiments and lacking evidence for inactivation through hypermethylation, these results indicate MAP3K7 as a gene for which haploinsufficency is substantially tumorigenic.

Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches.

The nature of cell-associated carbohydrates in the human intestine that may mediate transepithelial transport of bacterial and dietary lectins and their processing by the lymphoid cells of Peyers patches is not known. Because the cell surface carbohydrate receptors for lectins may vary in different species, the glycoconjugates of human and mouse follicle-associated epithelium and gut-associated lymphoid tissue were compared. A panel of 27, mainly recently isolated, lectins were used to identify glycoconjugate expression in M-cells, enterocytes, goblet cells, lymphocytes and macrophages in mouse and human intestine. Mouse M-cells were exclusively labelled by fucose-specific lectins but in human follicle-associated epithelium no distinct M-cell staining pattern was observed. In the human Peyers patches,Bryonia dioica lectin bound selectively to paracortical T-lymphocytes andChelidonium majus lectin to germinal centre B-cells. Certain mannose-specific lectins (Galanthus nivalis, Hippeastrum hybrid) stained the tingible body macrophages in the germinal centre of human Peyers patches but labelled the macrophages in the paracortical T-cell region of the mouse. The results indicate distinct differences in glycosylation between mouse and human Peyers patches and their associated lymphoid cells. When considering cell surface glycoconjugates as target molecules for the gut immune system, care has to be taken to choose the appropriate lectin for each species.