Alexander von Ruecker

Surgical stress induces a shift in the type-1/type-2 T-helper cell balance, suggesting down-regulation of cell-mediated and up-regulation of antibody-mediated immunity commensurate to the trauma*

BACKGROUND Measuring serum cytokines, pituitary hormones, or acute phase proteins during or after surgery is not an optimal method for quantifying the impact of surgical procedures. In an effort to assess surgical stress by means of the immune response, we focused on changes in cell-mediated and antibody-mediated immunity as illustrated by the type 1/type 2 T-helper (Th1/Th2) cell balance. The sensitivity of this approach was evaluated by comparing laparoscopic and conventional cholecystectomy (LCE, CCE). METHODS In a pragmatic prospective study 43 patients with symptomatic cholelithiasis were operated on either by LCE (n = 25) or CCe (n = 18). Blood sampling was done 24 hours before surgery, immediately before incision, and 2, 24, and 48 hours after surgery. Cell surface markers and cytokine production were used to characterize the Th1/Th2 balance and were measured by means of flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS Activation of Th2 cells evokes the production and secretion of interleukin-4 (IL-4), which up-regulates the expression of immunoglobulin E receptors (Fo epsilon RII, CD23) on B cells. Phytohemagglutinin-induced IL-4 production in freshly isolated peripheral blood mononuclear cells from patients increased more after CCE than LCE (IL-4, +41% versus +17%; p < 0.05). Also the expression of CD23 on B cells was higher after CCE than LCE (+146% versus +63%; P < 0.01). CD30, a membrane molecule that belongs to the tumor necrosis factor receptor superfamily and probably is an important indicator of Th2 activity, was more evaluated on T cells from patients who underwent CCE. The Th1 response, characterized by phytohemagglutinin-induced IFN-gamma secretion in peripheral blood mononuclear cells and up-regulation of human leukocyte antigen-DR expression on monocytes, was lower after CCE than after LCE. CONCLUSIONS This study shows that surgical stress induces a shift in the Th1/Th2 balance toward Th2, suggesting that cell-mediated immunity is down-regulated and antibody-mediated immunity is up-regulated after surgery. The evaluation of this shift may be clinically meaningful and help quantify even less invasive surgical procedures. When comparing CCE and LCE in this not strictly randomized study, we found LCE to be the less stressful procedure.

Circulating microRNAs (miRNA) in Serum of Patients With Prostate Cancer

OBJECTIVES To analyze circulating microRNAs (miRNA) in serum as non-invasive biomarker in patients with localized prostate cancer (PCA), benign prostate hyperplasia (BPH) and healthy individuals (HI). METHODS Total RNA was isolated from serum samples and the circulating levels of different RNA species (miRNA, miR-16; small nuclear RNA, RNU1A-1; messenger RNA, HPRT1), as well as of 4 oncogenic miRNAs (miR-26a, miR-32, miR-195, miR-let7i), were determined using a quantitative real-time polymerase chain reaction. We also evaluated miRNA levels in a second cohort of 10 PCA patients in cancer/nonmalignant tissue, and pre- and post-prostatectomy serum samples. RESULTS The levels of miR-16 and RNU1A-1 were reliably measured, whereas HPRT1 levels were often below the detection limit of our assay. Circulating oncogenic miRNA levels were different, and especially the miR-26a level allowed sensitive (89%) discrimination of PCA and BPH patients at a moderate specificity (56%; area under the curve [AUC]: 0.703); the analysis of oncogenic miRNAs in combination increased the diagnostic accuracy (sensitivity: 78.4%; specificity: 66.7%; AUC: 0.758). Despite the low number of patients limiting the statistical power of the study, we observed correlations with clinical-pathologic parameters: miR-16, miR-195, and miR-26a were significantly correlated with surgical margin positivity; miR-195 and miR-let7i were significantly correlated with the Gleason score. Tissue miRNA levels were correlated with preprostatectomy miRNA levels in serum, and serum miRNA decreased after prostatectomy, thereby indicating tumor-associated release of miRNA. CONCLUSIONS Tumor-associated miRNAs in serum allow noninvasive discrimination of PCA and BPH.

Allogeneic dendritic cells fused with tumor cells: preclinical results and outcome of a clinical phase I/II trial in patients with metastatic renal cell carcinoma.

Therapeutic vaccination with dendritic cells (DC) can lead to tumor regression in animal models and has shown promising results in the first clinical trials of metastatic renal cell carcinoma and malignant melanoma. In vitro data and results of a clinical phase I/II trial using DC tumor fusions in patients with progressive metastatic renal cell carcinoma are presented here. In addition to toxicity and feasibility, complex immune monitoring was a point of interest. DC precursor cells were obtained from the peripheral blood mononuclear cells (PBMCs) of healthy donors and were fused with either allogeneic (8 patients) or autologous (4 patients) renal tumor cells. In total, 12 patients with progressive metastatic renal cell carcinoma were treated with an average of 2.8 x 10(7) tumor cells fused with 1.8 x 10(7) DC each administered on days 0, 28, and 56 intradermally. Fusion efficacy for the tumor cells used was 14.3% +/- 7.8%. Cell viability was 59.8% +/- 6.8% after fusion and irradiation. We observed no adverse effects and no difference in clinical outcome between the allogeneic and the autologous treatment. Eight patients remained in a progressive disease state and four patients in a stable disease state. T-cell immunity was carefully monitored before, during, and after treatment. Delayed-type hypersensitivity (DTH) reaction using tumor cells was positive after treatment in 7 of 12 patients, 2 of whom were found to have stable disease. An increase in the reactivity against recall antigens was seen in most patients. Interestingly, cytotoxicity of peripheral blood lymphocytes (PBLs) against renal cell carcinoma cells increased during treatment as well as the percentage of interferon-gamma-secreting cells. This effect was significantly enhanced within the group that had stable disease. The lack of adverse effects together with positive immunologic signs justifies further investigation of this novel therapeutic approach. Further studies are necessary to test for clinical effectiveness in patients with tumors, especially those with less advanced disease.

Global Levels of Histone Modifications Predict Prostate Cancer Recurrence

Epigenetic alterations such as DNA methylation and histone modifications play important roles in carcinogenesis. It was reported that global histone modification patterns are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone lysine (HxKy) and histone acetyl (HxAc) modifications in prostate tissue.

Diagnostic and Prognostic Information in Prostate Cancer with the Help of a Small Set of Hypermethylated Gene Loci

Purpose: Our study was designed to evaluate promoter CpG island hypermethylation in the diagnosis and prognosis of prostate cancer. Experimental Design: Primary prostate cancers from 53 patients, pelvic lymph nodes, noncancerous prostate tissues, and prostate cell lines were analyzed. Real-time methylation-specific PCR was used to identify CpG island hypermethylation at five promising gene loci (i.e., GSTP1, APC, PTGS2, MDR1, and RASSF1a). Results: At three gene loci (GSTP1, APC, and PTGS1) and CpG island, hypermethylation was highly prevalent in prostate cancers (71-91%), and analysis of receiver operator curves showed that hypermethylation at these three gene loci can distinguish between prostate cancer and noncancerous prostatic tissue (i.e., benign hyperplasia) with a sensitivity of 71.1% to 96.2% and a specificity of 92.9% to 100%. Using sensitive SYBR green methylation-specific PCR technology, we observed a respective 28% and 71% hypermethylation rate at the RASSF1a and MDR1 loci in benign prostate hyperplasia, which may represent early nonaggressive carcinogenesis. Methylation characteristics in prostate cancer metastases (i.e., pelvic lymph nodes) were comparable to the respective primary cancer. Statistical analysis showed no correlation between the methylation status of a single gene locus and clinicopathologic variables (e.g., preoperative prostate specific antigen levels, Gleason score, capsular penetration, involvement of seminal vesicle, and age). In contrast, the methylation of two (GSTP1/APC; GSTP1/PTGS2) or three (GSTP1/APC/PTGS2) gene loci correlated with prognostic indicators (i.e., pathologic stage, extraprostatic extension, and Gleason score, but not with prostate specific antigen levels). Conclusions: Our data suggest that the evaluation of DNA hypermethylation at three gene loci (i.e., GSTP1, APC, and PTGS2) is of diagnostic and prognostic value in prostate cancer.

CpG island hypermethylation at multiple gene sites in diagnosis and prognosis of prostate cancer.

OBJECTIVES CpG island hypermethylation causes gene silencing and could be decisive in prostate carcinogenesis and progression. We investigated its role at multiple gene sites during prostate carcinogenesis. METHODS A quantitative, methylation-specific polymerase chain reaction was used to analyze the hypermethylation patterns at nine gene loci (Annexin2, APC, EDNRB, GSTP1, PTGS2, MDR1, RARbeta, Reprimo, and TIG1) in 80 patients with prostate cancer (PCa) and 26 patients with benign prostatic hyperplasia (BPH). RESULTS Hypermethylation was more frequent in PCa than in BPH tissues (EDNRB, 100% versus 88%; TIG1, 96% versus 12%; RARbeta, 95% versus 35%; GSTP1, 93% versus 15%; APC, 80% versus 50%; MDR1, 80% versus 31%; PTGS2, 68% versus 15%; Reprimo, 59% versus 19%; and Annexin2, 4% versus 0%). TIG1 and GSTP1 hypermethylation distinguished between PCa and BPH with a specificity of greater than 85% and sensitivity of greater than 93%. Hypermethylation at a single gene locus did not correlate with any clinicopathologic variables. In contrast, hypermethylation at two genes (eg, APC and TIG1, APC and GSTP1, APC and PTGS2, APC or MDR, GSTP1 or PTGS2) correlated significantly with the pathologic stage and/or Gleason score (P = 0.033 to 0.045). Hypermethylation at APC and Reprimo, as well as DNA hypermethylation at more than five genes, correlated significantly with the rate of prostate-specific antigen recurrence after radical prostatectomy (P = 0.0078 and P = 0.0074, respectively). CONCLUSIONS Our results have confirmed that the hypermethylation patterns are helpful in the diagnosis and prognosis of PCa. Increases in CpG island hypermethylation at multiple gene sites occur during PCa progression and indicate early biochemical recurrence after radical prostatectomy.

Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial

Abstract. In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95×106 DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4×106 DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.

Noncancerous PTGS2 DNA fragments of apoptotic origin in sera of prostate cancer patients qualify as diagnostic and prognostic indicators

Our study was designed to evaluate Cell‐Free DNA in sera of prostate cancer (PCA) patients as a useful biomarker. Real‐time PCR was used to amplify a <200 bp PTGS2 DNA fragment that biochemically characterizes apoptosis and a larger >250 bp Reprimo DNA fragment that defines mostly other cell death entities. The apoptosis index (AI) expresses the ratio of PTGS2 to Reprimo DNA fragments. GSTP1 hypermethylation was assessed to evaluate the amount of tumor‐derived DNA. We analyzed serum of 216 patients (168 PCA; 5 incidental PCA; 42 benign prostatic hyperplasia (BPH); 11 healthy individuals). Distinctly elevated concentrations of PTGS2 fragments were detected in PCA compared to BPH and healthy individuals (median: 70.2, 10.5 and 7.1 ng/ml, respectively; p < 0.0001). The AI was significantly increased in PCA vs. BPH patients and healthy individuals (6.01 vs. 1.54 and 0.84 respectively; p = 0.002–0.0001). GSTP1 hypermethylation was only present in a small percentage (mean 1.92%) of circulating DNA. Concentrations of apoptotic PTGS2 fragments discriminated sensitively (88%) and specifically (64%) between BPH and PCA, whereas the AI was more specific (82%) but less sensitive (70%). The AI correlated with histological grading (p = 0.044). Kaplan–Meier analysis for a subset of 124 patients revealed a significant correlation between apoptotic PTGS2 fragments or the AI and PSA recurrence following radical prostatectomy (p = 0.0395–0.0482). In conclusion, circulating PTGS2 fragments of apoptotic origin and the AI are promising serum biomarkers for the diagnosis and prognosis of PCA. We suggest that cancer‐induced apoptosis of peripheral noncancerous tissues is relevant in many malignancies.

Analysis of serum microRNAs (miR-26a-2*, miR-191, miR-337-3p and miR-378) as potential biomarkers in renal cell carcinoma

INTRODUCTION Emerging evidence suggest that microRNAs could serve as non-invasive biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC). MATERIALS AND METHODS Serum RNA was isolated from patients with clear cell RCC (ccRCC) and non-malignant disease; an artificial microRNA (cel-miR-39) was spiked-in prior the isolation procedure to control isolation efficiency. The levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 in serum were determined using quantitative real-time PCR; the microRNA levels were normalized to cel-miR-39. RESULTS First, miR-26a-2*, miR-191, miR-337-3p and miR-378 were quantified in serum of each 25 patients with ccRCC and non-malignant disease. The level of miR-378 was significantly increased in ccRCC patients, and thus chosen for validation. The analysis of miR-378 in the validation cohort with 117 RCC patients and 123 control subjects did not confirm a different level of miR-378. Also, miR-378 was not correlated to pT-stage, lymph node/distant metastasis, vascular invasion and Fuhrman grade. CONCLUSIONS The analysis of circulating serum levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 is unlikely to provide helpful diagnostic/prognostic information in RCC patients.

Prognostic relevance of global histone H3 lysine 4 (H3K4) methylation in renal cell carcinoma

Epigenetic alterations play an important role in carcinogenesis. Recent studies suggested that global histone modifications are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone H3 lysine 4 mono‐methyl (H3K4me1), ‐di‐methyl (H3K4me2) and ‐trimethyl (H3K4me3) patterns in renal cell carcinoma (RCC) using a tissue microarray with 193 RCC (including 142 clear cell, 31 papillary, 10 chromophobe and 10 sarcomatoid RCC) and 10 oncocytoma specimens: H3K4me3 staining was more intense in papillary RCC, whereas H3K4me1 and H3K4me2 were similar in the diverse RCC subtypes. H3K4me2 and H3K4me3 levels were increased in oncocytoma. H3K4me1‐3 levels were inversely correlated with Fuhrman grading, pT stage, lymph node involvement and distant metastasis. Progression‐free survival and cancer‐specific survival were shorter in patients with low levels of H3K4me1‐3 in the univariate analysis, but we did not observe a significant correlation of a single modification in a multivariate model, which also included the established prognostic parameters TNM‐stage and Fuhrman grade. In comparison, the H3K4me score, which combined staining levels of the H3K4 modifications, was an independent predictor of RCC progression‐free survival. Our study on H3K4 methylation supports the concept of global histone modifications as potential cancer prognosis markers.