SATB1, genomic instability and Gleason grading constitute a novel risk score for prostate cancer.

Current prostate cancer risk classifications rely on clinicopathological parameters resulting in uncertainties for prognostication. To improve individual risk stratification, we examined the predictive value of selected proteins with respect to tumor heterogeneity and genomic instability. We assessed the degree of genomic instability in 50 radical prostatectomy specimens by DNA-Image-Cytometry and evaluated protein expression in related 199 tissue-microarray (TMA) cores. Immunohistochemical data of SATB1, SPIN1, TPM4, VIME and TBB5 were correlated with the degree of genomic instability, established clinical risk factors and overall survival. Genomic instability was associated with a GS ≥ 7 (p = 0.001) and worse overall survival (p = 0.008). A positive SATB1 expression was associated with a GS ≤ 6 (p = 0.040), genomic stability (p = 0.027), and was a predictor for increased overall survival (p = 0.023). High expression of SPIN1 was also associated with longer overall survival (p = 0.048) and lower preoperative PSA-values (p = 0.047). The combination of SATB1 expression, genomic instability, and GS lead to a novel Prostate Cancer Prediction Score (PCP-Score) which outperforms the current D'Amico et al. stratification for predicting overall survival. Low SATB1 expression, genomic instability and GS ≥ 7 were identified as markers for poor prognosis. Their combination overcomes current clinical risk stratification regimes.

Claudin-1 expression in cervical cancer.

Claudin-1 is a tight junction protein that has been demonstrated to be involved in tumorigenesis and tumor progression in various types of solid tumors. In the present study, the protein expression of claudin-1 in squamous cervical cancer tissues obtained from 106 patients was analyzed by immunohistochemistry. In addition, the grade of claudin-1 expression was analyzed for associations with certain clinicopathological parameters. A significant overexpression of claudin-1 was detected in the tumor cells, when compared with that in the peritumoral stroma. There was no significant association between claudin-1 expression and FIGO stage, tumor size, grading or the appearance of distant metastases. Cervical cancer patients scoring positive for claudin-1 protein expression tended to exhibit more lymph node metastasis (28.3%), compared with claudin-1-negative patients (7.1%). Regarding overall survival, the results of the present study suggest a better prognosis for claudin-1-negative patients. In order to elucidate whether claudin-1 overexpression has a significant prognostic impact on squamous cervical cancer, further studies are required.

EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer.

BACKGROUND: While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. RESULTS: Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. MATERIALS AND METHODS: Fresh frozen tissue of UCC (n = 10) matched with SCC (n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. CONCLUSIONS: This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort.

Expression of cyclooxygenase-2 in cervical cancer is associated with lymphovascular invasion.

Cyclooxygenase-2 (COX-2) is associated with carcinogenesis and tumor progression. The current study analyzed the effect of COX-2 expression in patients with invasive squamous cervical cancer. Tissue samples from 123 cervical cancer patients were collected for a retrospective analysis using immunohistochemistry (IHC) with an antibody against COX-2. The clinical and survival data of the patients were analyzed. Positive staining for COX-2 (defined as an immunoreactivity score of ≥4) was detected in 28 patients (23%), with significantly higher percentages of staining in tumor cells compared with peritumoral stroma cells (P<0.001). COX-2 expression was significantly associated with lymphovascular space invasion (LVSI; P=0.017). The association of COX-2 expression with LVSI suggests a possible effect of COX-2 on tumor progression in cervical cancer. Further studies including larger patient collectives are required in order to perform analyses of clinical subgroups and patient survival.

Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks.

BACKGROUND: Biobanks are becoming increasingly important for assessment of disease risk as well as identification and validation of new diagnostic biomarkers and druggable targets. The validity of data obtained from biobanks is critically limited by the biomaterial quality of the biological samples. External quality assessment (EQA) programs suitable to comprehensively measure the biomaterial quality in archived materials are currently lacking. We report on quantitative assay designs for the analysis of both structural and functional integrity of DNAs that were applied in a first pilot EQA within the priority program on tumor tissue biobanking funded by the German Cancer Aid. METHODS: Participating biobanks isolated DNAs from a standardized set of 10 samples comprising sections of four different formalin-fixed paraffin-embedded tissues using their standard operating procedures. Isolated DNAs and analytical results were returned and analyzed centrally for nucleic acids yield, purity, fragmentation and amplificability at a quantitative level using dedicated assay designs. RESULTS: The amount of extracted DNA varied in isolates ranging between 1.5 µg and 25.8 µg. Quantification of DNA fragmentation and amplificability allowed to highlight considerable discrepancies in DNA quality. Amplicons yielded from the isolates of these identical EQA samples ranged from 105 to 411 bp suggesting differences between residual inhibitors of downstream enzymatic reactions. CONCLUSIONS: The quality of extraction of bioanalytes from biomaterial archives is heterogeneous even for stable biomolecules like DNA isolated with highly standardized methods. EQAs are appropriate tools to uncover strengths and weaknesses in biobanks in a systematic fashion. Biomaterial integrity is insufficiently reflected by standard methods, but needs to be assessed to improve biobank interoperability. Finally, our results also point towards the problem of measuring the quality of more delicate biomolecules like proteins or metabolites.

The impact of pre-analytical conditions on the serum proteome: heat-stabilization versus nitrogen storage.

CONTEXT: Biological material reflecting the in vivo composition of markers provides a high potential for biomarker discovery. OBJECTIVE: We compared the serum proteome following heat- and nitrogen-preservation, with and without subsequent storage at room temperature. MATERIALS AND METHODS: Serum samples were collected, treated and analysed by two-dimensional gel electrophoresis. Protein spots were identified and confirmed by two mass spectrometry approaches (MALDI & ESI) and subjected to Ingenuity Pathway Analysis. RESULTS: We revealed 24 differentially expressed proteins (p ≤ 0.05) between nitrogen and heat preservation, and 87 between nitrogen and heat preservation with subsequent storage for 120 h at room-temperature. Mass spectrometry identified 25 polypeptides. Pathway analysis resulted in networks maintaining Cellular Assembly and Organization, Movement and Maintenance. CONCLUSION: Heat-stabilization does not substantially change the short-term proteome composition of serum compared with nitrogen treatment. However, heat-stabilization alone seems insufficient for long-term sample preservation for serum samples. We identified transthyretin and apolipoprotein A-IV as sample quality markers.

The "North German Tumor Bank of Colorectal Cancer": status report after the first 2 years of support by the German Cancer Aid Foundation.

PURPOSE: Research projects and clinical trials strongly rely on high-quality biospecimens which are provided by biobanks. Since differences in sample processing and storage can strongly affect the outcome of such studies, standardization between biobanks is necessary to guarantee reliable results of large, multicenter studies. The German Cancer Aid Foundation (Deutsche Krebshilfe e.V.) has therefore initiated the priority program "tumor tissue banks" in 2010 by funding four biobank networks focusing on central nervous system tumors, melanomas, breast carcinomas, and colorectal carcinomas. The latter one, the North German Tumor Bank of Colorectal Cancer (ColoNet) is managed by surgeons, pathologists, gastroenterologists, oncologists, scientists, and medical computer scientists. METHODS AND RESULTS: The ColoNet consortium has developed and harmonized standard operating procedures concerning all biobanking aspects. Crucial steps for quality assurance have been implemented and resulted in certification according to DIN EN ISO 9001. A further achievement is the construction of a web-based database for exploring available samples. In addition, common scientific projects have been initiated. Thus, ColoNet's repository will be used for research projects in order to improve early diagnosis, therapy, follow-up, and prognosis of colorectal cancer patients. Apart from the routine sample storage at -170 °C, the tumor banks' unique characteristic is the participation of outpatient clinics and private practices to further expand the sample and clinical data collection. CONCLUSION: The first 2 years of funding by the German Cancer Aid Foundation have already led to a closer scientific connection between the participating institutions and to a substantial collection of biospecimens obtained under highly standardized conditions.

Aneuploidy and elevated CEA indicate an increased risk for metachronous metastasis in colorectal cancer.

PURPOSE: Presently, no markers exist to predict metachronous metastasis at the time a primary colorectal cancer is diagnosed. While aneuploidy indicates poor survival prognosis and elevated carcinoembryonic antigen (CEA) levels the presence of recurrent disease, the predictive value of both markers regarding imminent metachronous metastases is unclear. METHODS: Sixty patients with distant recurrence throughout a 5-year follow-up (TM+) were randomly chosen and 60 patients without metastasis matched to this cohort (TM-). In addition, an enlarged collective (n = 217; n TM+ = 85, n TM- = 132) with median follow-up of 79.2 months was assessed by logistic regression regarding metachronous metastases. Univariate and stepwise regression analyses included clinicopathological characteristics, preoperative CEA levels and aneuploidy assessed by DNA image cytometry. RESULTS: The matched-pair collective showed aneuploidy in 71.1 % (TM-) and 85.0 % (TM+; p = 0.076), and elevated CEA in 24.5 % (TM-) and 52.2 % [TM+; odds ratio (OR), 3.414; p = 0.007]. The enlarged collective presented aneuploidy in 71.2 % (TM-) and 83.5 % (TM+; OR 2.050, p = 0.038), and elevated CEA in 28.6 % (TM-) and 48.9 % (TM+; OR 2.391, p = 0.020). Elevated CEA and aneuploidy did not show any association (p = 0.919). In contrast, logistic regression analyses demonstrated that besides increased T category (OR 1.745, p = 0.019), both elevated CEA level (OR 2.633, p = 0.015) and aneuploidy (OR 1.929, p = 0.058) were independent predictive markers for metachronous metastasis. CONCLUSIONS: Our data show that aneuploidy and elevated CEA levels besides increased T category could serve for individual risk assessment to predict metachronous metastases. The fact that still aneuploidy missed the significance level by a small margin emphasizes the need for larger validation studies.

Effects of the CC chemokine receptor 2 in mice deficient for the myelin protein zero (P0).

In myelin protein zero (P0)-deficient mice immune cells are critically involved in the pathogenesis of a primary genetic disease. Previously it has been shown that the chemokine CCL2 affects the functional properties of endoneurial macrophages in heterozygous P0 mice. The aim of the present study was to characterize the role of the CCL2-receptor CCR2 in the pathogenesis of the neuropathy in P0 deficient mice. In demyelinating nerves of heterozygous P0 mice (P0+/-) CCR2-deficiency did not affect the number of endoneurial macrophages; there was a trend towards a higher number of activated macrophages. CCR2-deficiency resulted in an increased nerve demyelination. In dysmyelinating nerves of homozygous P0 mice (P0-/-), CCR2-deficiency led to a significant decrease of endoneurial macrophages but did not affect axonal degeneration. There was no effect of CCR2 on T-lymphocytes in both disease models. Our data confirm a functional role of the CCR2 receptor in the examined models of hereditary neuropathies. In P0+/- mutants CCR2 decreases macrophage activation and is protective against demyelination, whereas in P0-/- mice it increases the accumulation of endoneurial macrophages.