Mahmoud Abbas

Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.

DNA methylation biomarkers predict progression-free and overall survival of metastatic renal cell cancer (mRCC) treated with antiangiogenic therapies.

VEGF-targeted therapy increases both the progression-free (PFS) and overall survival (OS) of patients with metastasized renal cell cancer (mRCC). Identification of molecular phenotypes of RCC could improve risk-stratification and the prediction of the clinical disease course. We investigated whether gene-specific DNA hypermethylation can predict PFS and OS among patients undergoing anti-VEGF-based therapy. Primary tumor tissues from 18 patients receiving targeted therapy were examined retrospectively using quantitative methylation-specific PCR analysis of CST6, LAD1, hsa-miR-124-3, and hsa-miR-9-1 CpG islands. PFS and OS were analyzed for first-line and sequential antiangiogenic therapies using the log rank statistics. Sensitivity and specificity were determined for predicting first-line therapy failure. Hypermethylation of CST6 and LAD1 was associated with both a shortened PFS (log rank p = 0.009 and p = 0.004) and OS (p = 0.011 and p = 0.043). The median PFS observed for the high and low methylation groups of CST6 and LAD1 was 2.0 vs.11.4 months. LAD1 methylation had a specificity of 1.0 (95% CI 0.65–1.0) and a sensitivity of 0.73 (95% CI 0.43–0.90) for the prediction of first-line therapy. CST6 and LAD1 methylation are candidate epigenetic biomarkers showing unprecedented association with PFS and OS as well as specificity for the prediction of the response to therapy. DNA methylation markers should be considered for the prospective evaluation of larger patient cohorts in future studies.

Genome-Wide Investigation of Multifocal and Unifocal Prostate Cancer — Are They Genetically Different?

Prostate cancer is widely observed to be biologically heterogeneous. Its heterogeneity is manifested histologically as multifocal prostate cancer, which is observed more frequently than unifocal prostate cancer. The clinical and prognostic significance of either focal cancer type is not fully established. To investigate prostate cancer heterogeneity, the genetic profiles of multifocal and unifocal prostate cancers were compared. Here, we report observations deduced from tumor-tumor comparison of copy number alteration data of both focal categories. Forty-one fresh frozen prostate cancer foci from 14 multifocal prostate cancers and eight unifocal prostate cancers were subjected to copy number variation analysis with the Affymetrix SNP 6.0 microarray tool. With the investigated cases, tumors obtained from a single prostate exhibited different genetic profiles of variable degrees. Further comparison identified no distinct genetic pattern or signatures specific to multifocal or unifocal prostate cancer. Our findings suggest that samples obtained from multiple sites of a single unifocal prostate cancer show as much genetic heterogeneity and variability as separate tumors obtained from a single multifocal prostate cancer.

Neurofilament Heavy polypeptide CpG island methylation associates with prognosis of renal cell carcinoma and prediction of antivascular endothelial growth factor therapy response

Neurofilament Heavy polypeptid (NEFH) belongs to the group of type IV intermediate filament proteins. DNA methylation of the NEFH promoter and loss of expression have previously been shown to activate the AKT/β‐catenin pathway in tumor cells. When identifying hypermethylation of the NEFH CpG island (CGI) in renal cell cancer (RCC) we asked whether methylation could provide clinical or prognostic information for RCC and/or predict therapy response in patients with metastatic RCC (mRCC) undergoing antiangiogenic therapy. Relative methylation of the NEFH CGI was analyzed in 132 RCC samples and 83 paired normal tissues using quantitative methylation‐specific PCR. Results were statistically compared with tumor histology, clinicopathological parameters, progression‐free survival (PFS) as well as with overall survival (OS) in a subset of 18 mRCC patients following antiangiogenic therapy regimens. The NEFH CGI methylation demonstrated a tumor‐specific increase (P < 0.001), association with advanced disease (P < 0.001), and distant metastasis (P = 0.005). Higher relative methylation was also significantly associated with a poor PFS (HR = 8.6, P < 0.001) independent from the covariates age, gender, diameter of tumors, state of advanced disease, and local and distant metastasis. Median OS following targeted therapy was 29.8 months for patients with low methylation versus 9.8 months for the group with high methylation (P = 0.028). We identified NEFH methylation as a candidate epigenetic marker for prognosis of RCC patients as well as prediction of anti‐vascular endothelial growth factor‐based therapy response.

High reproducibility of adhesion formation in rat with meso-stitch approximation of injured cecum and abdominal wall.

Objective: Peritoneal adhesions following surgery are a common, serious pathology with severe complications. Appropriate animal adhesion models are essential for the assessment of adhesion preventing medical devices. This study introduces a variation of an established rat model in which highest degree adhesions are induced with excellent reproducibility (OPAM = optimized peritoneal adhesion model). Thus, this model seems to be eligible to study effects of adhesion preventing devices. Methods: 24 Lewis male rats were divided into four groups (OPAM, WSFX, sham-OPAM, sham-WSFX). The OPAM technique comprised cecal abrasion, creation of an abdominal wall defect plus approximation of injured areas by a suture, which was compared to a setting of lesions without suture fixation (WSFX). All rats were sacrificed at day 7. Macroscopic and histopathological evaluations were performed. Results were statistically analyzed using ANOVA and Dunnetts test. Results: In OPAM rats macroscopic analyses revealed a 90% incidence adhesion of cecum to the abdominal wall, all adhesions imposing as complete agglutination. In WSFX animals incidence of adhesions formation was 75%, while in both sham groups there were no adhesions at all. Histology showed the structure of adhesions with merged smooth muscle of colon and skeletal muscle of abdominal wall in all cases. Conclusion: OPAM technique provides adhesions of injured areas with a better probability than with conventional methods. All OPAM adhesions impressed as highest degree adhesions, i.e. agglutination. Due to high reproducibility in incidence and extend of adhesion formation, the OPAM is recommended for testing of adhesion prevention medical devices.

Galectin-1 and Galectin-3 mRNA expression in renal cell carcinoma

BackgroundGalectins are known to regulate cell differentiation and growth as well as cell adhesion and apoptosis. Galectins have been discussed as possible prognosticators for survival in renal cell cancer (RCC) and other urological tumors. They might also play an emerging role as possible new marker-proteins for RCC. In this study, we analyzed the expression of galectin-1 and galectin-3 mRNA in order to further investigate their clinical significance in RCC.MethodsTissue samples were obtained from 106 patients undergoing surgery for RCC. The expression of galectin-1 and galectin-3 mRNA in normal kidney and corresponding cancer tissue was analyzed using quantitative real time PCR. Differences in expression levels of paired tissue samples were assessed using paired two-sample tests. Associations of relative mRNA expression levels in tumor tissues with clinical findings were analyzed using univariate logistic regression.ResultsThe expression of galectin-1 (p < 0.001) and -3 (p < 0.001) mRNA were significantly higher in RCC when compared to the adjacent normal kidney tissue. For clear cell RCC, an association of male gender with higher galectin-1 and galectin-3 mRNA expression (p = 0.054, p = 0.034) was detected. For all RCCs, galectin-1 mRNA expression failed to show a significant association with advanced disease as well as a higher rate of lymph node metastases (p = 0.058, p = 0.059).ConclusionThe mRNA expression of galectin-1 and galectin-3 is significantly increased in RCC cancer tissue. The higher mRNA expression in tumor tissue of male patients raises the question of a functional connection between galectins and the higher prevalence of RCC in men. Associations with advanced disease might lead to new ways of identifying patients at higher risk of recurrent disease and might even facilitate early metastasectomy with curative intent.

Evaluation of 4DryField® PH as Adhesion Prevention Barrier Tested in an Optimized Adhesion Model in Rats.

Background: Adhesions due to pelvic/abdominal surgery are a common serious pathology possibly entailing severe complications. This study investigates the adhesion prevention capability of the novel starch-based agent 4DryField® PH, which together with saline solution forms a barrier gel. Herein, an optimized adhesion model (OPAM) inducing severe adhesions/agglutinations with high reproducibility was used. Methods: In 19 Lewis rats, a 1 × 2 cm abdominal wall defect was created, the peritoneum of the neighboring cecum was abraded, and both injured areas were approximated by suture. Rats were randomized to control (n = 10) or 4DryField PH treatment (n = 9) groups. Another 8 rats had sham surgery for safety assessment of 4DryField PH. At day 7, the quantity and quality of adhesions were assessed macro-/microscopically and evaluated statistically. Results: 4DryField PH treatment significantly reduced the incidence and severity of adhesions as verified by significantly improved adhesion scorings (0.4 vs. 4.5; 1.1 vs. 9). Histology revealed reconstitution of the cecum and abdominal wall including regeneration of the visceral/parietal peritoneum. In sham-operated rats, 4DryField PH did not induce adhesion formation. Conclusions: 4DryField PH gel was highly effective in preventing adhesions. Histologically, the injured cecum and abdominal wall regenerated well in the presence of 4DryField PH. Considering the severity of OPAM trauma, the potential of 4DryField PH to prevent adhesions can be rated excellent.

CMDX©-based single source information system for simplified quality management and clinical research in prostate cancer

BackgroundHistopathological evaluation of prostatectomy specimens is crucial to decision-making and prediction of patient outcomes in prostate cancer (PCa). Topographical information regarding PCa extension and positive surgical margins (PSM) is essential for clinical routines, quality assessment, and research. However, local hospital information systems (HIS) often do not support the documentation of such information. Therefore, we investigated the feasibility of integrating a cMDX-based pathology report including topographical information into the clinical routine with the aims of obtaining data, performing analysis and generating heat maps in a timely manner, while avoiding data redundancy.MethodsWe analyzed the workflow of the histopathological evaluation documentation process. We then developed a concept for a pathology report based on a cMDX data model facilitating the topographical documentation of PCa and PSM; the cMDX SSIS is implemented within the HIS of University Hospital Muenster. We then generated a heat map of PCa extension and PSM using the data. Data quality was assessed by measuring the data completeness of reports for all cases, as well as the source-to-database error. We also conducted a prospective study to compare our proposed method with recent retrospective and paper-based studies according to the time required for data analysis.ResultsWe identified 30 input fields that were applied to the cMDX-based data model and the electronic report was integrated into the clinical workflow. Between 2010 and 2011, a total of 259 reports were generated with 100% data completeness and a source-to-database error of 10.3 per 10,000 fields. These reports were directly reused for data analysis, and a heat map based on the data was generated. PCa was mostly localized in the peripheral zone of the prostate. The mean relative tumor volume was 16.6%. The most PSM were localized in the apical region of the prostate. In the retrospective study, 1623 paper-based reports were transferred to cMDX reports; this process took 15 ± 2 minutes per report. In a paper-based study, the analysis data preparation required 45 ± 5 minutes per report.ConclusionscMDX SSIS can be integrated into the local HIS and provides clinical routine data and timely heat maps for quality assessment and research purposes.

The presence of positive surgical margins in patients with organ-confined prostate cancer results in biochemical recurrence at a similar rate to that in patients with extracapsular extension and PSA≤10 ng/ml

PURPOSES We investigated whether patients with organ-confined prostate cancer (PCa) and positive surgical margins (SMs) had a similar biochemical recurrence (BCR) risk compared with patients with pT3a and preoperative prostate-specific antigen (PSA) levels ≤ 10ng/ml. Furthermore, we examined the effects of incorporating SM status, Gleason score (Gls), and preoperative PSA level into the discrimination accuracy of the current tumor node metastasis-staging system. MATERIALS AND METHODS We analyzed 863 PCa patients treated with radical prostatectomy from 1999 to 2008. Only individuals with pT2N0 or pT3N0, without neoadjuvant or adjuvant therapy, were included. We performed chi-square automatic interaction detection analysis to generate a classification model for predicting BCR by analyzing interactions between age at surgery, SM status, Gls, PSA, and tumor stage, tumor volume and relative tumor volume. Cox regression analyses tested the relationship between SM status and BCR rate after stratification according to T-stage and the novel classification. The predictive and discrimination accuracy of the current T-stage and of the classification model was quantified with time-dependent receiver operating characteristics and integrated discrimination improvement. The topographical association between extracapsular extension of PCa and positive SM was analyzed in patients with pT3aR1 using a computational reconstruction diagram of the prostate. RESULTS The chi-square automatic interaction detection analysis found interactions among pT Stage, SM status, PSA and Gls and generated a classification model for BCR prediction: pT2R0, pT2R1, pT3a PSA ≤ 10 ng/ml, pT3a PSA>10 ng/ml and pT3b. Men with pT2R1 had a shorter time to BCR compared with men with pT3a-PSA ≤ 10 ng/ml (P<0.0001). Gls≥7a was correlated with a poorer BCR rate than Gls≤7a in men with pT2R1 or pT3a PSA ≤ 10 ng/ml (P = 0.012). The rank order (highest to lowest) for the risk of developing BCR was pT3b>pT2R1/pT3a-PSA>10 ng/ml>pT2R1/pT3a PSA ≤ 10 ng/ml>pT2R0 (P<0.0001). Discrimination accuracy gains were observed when PCa was stratified according to the novel classification (P<0.0001). A topographical association between extracapsular extension and positive SM was found in patients with pT3aR1 (P = 0.01). CONCLUSION Patients with pT2R1 develop a similar BCR risk to that of patients with pT3a PSA ≤ 10 ng/ml. Gls≥7b is associated with a high BCR risk in these patient groups. Including SM status, PSA, and Gls in pT stage appears to improve prognostic stratification in patients with PCa.

GATA5 CpG island hypermethylation is an independent predictor for poor clinical outcome in renal cell carcinoma

Transcriptional inactivation and CpG island (CGI) methylation of GATA transcription factor family members GATA3 and GATA5 have been reported for a few types of human cancer. Whether high-density CGI methylation of GATA3 or GATA5 is associated with the clinical course of patients with renal cell cancer (RCC) has not been clarified. Quantitative methylation-specific PCR assays were carried out to analyze 25 tumor cell lines including 6 RCC lines and 119 RCC and 87 adjacent normal tissues for the presence of densely methylated sequences. Methylation values were statistically compared with clinicopathological and recurrence-free survival (RFS) data for patients. Comparison of GATA3 and GATA5 methylation in different tumor cell lines revealed a marker-specific methylation characteristic with high and frequent signals for both methylation marks in RCC lines. GATA3 and GATA5 CGI relative methylation levels were found to be strongly associated with the state of metastasis (P=0.003 and P<0.001, respectively) and advanced disease (P=0.024 and P<0.001, respectively). Moreover, an independent decrease in RFS in Cox proportional hazard analysis was found for tumors exhibiting high GATA5 methylation (P<0.001, hazard ratio, 19.3; 95% confidence interval, 4.58–81.6). Epigenetic alterations in GATA family members may be associated with aggressive tumor phenotypes in RCC, and in the case of GATA5, may serve as a new independent molecular marker for aggressiveness and disease progression.