Gisela Walgenbach-Brünagel

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.

Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer, bladder cancer and renal cell carcinoma.

Objectives:  To identify an appropriate reference gene for the analysis of circulating micro‐ribonucleic acid in patients with urological malignancies.

Circulating mitochondrial DNA in serum: A universal diagnostic biomarker for patients with urological malignancies ☆

OBJECTIVE Cell-free circulating mitochondrial DNA (mtDNA) has been proposed as universal diagnostic and prognostic biomarker in cancer patients. PATIENTS AND METHODS Cell-free DNA was isolated from 1 ml serum from patients with bladder cancer (BCA, n = 84), renal cell carcinoma (RCC, n = 33), and prostate cancer (CaP, n = 23), and compared with healthy individuals (n = 79). Quantitative real-time PCR was used to analyze the levels of a 79 bp (mtDNA-79), and 220 bp (mtDNA-220) fragment of the mitochondrial specific 16S-RNA. The mitochondrial DNA integrity (mtDNA-integrity) was defined as ratio of mtDNA-220 to mtDNA-79 fragments. RESULTS In healthy controls, mtDNA-79 levels were increased in male volunteers; mtDNA-230 levels and mtDNA-integrity were correlated with age. Neither mtDNA levels nor mtDNA-integrity were correlated with age or gender in cancer patients. Circulating mtDNA-79 (median 8.75 × 10(6) vs. 0.43 × 10(6) copies/ml) and mtDNA-230 (8.11 × 10(6) vs. 0.27 × 10(6) copies/ml) levels were significantly increased in cancer patients and allowed sensitive (84%) and specific (97%) discrimination from healthy controls. mtDNA levels were unequally distributed among the different cancer entities (mtDNA-79: BCA 9.54 × 10(6) vs. RCC 6.69 × 10(6) vs. CaP 4.48 × 10(6) copies/ml; mtDNA-230: BCA 9.78 × 10(6) vs. RCC 6.74 × 10(6) vs. CaP 1.94 × 10(6) copies/ml). The mtDNA-integrity was increased in RCC and BCA patients compared to control subjects and CaP patients. Serum mtDNA-integrity was correlated with pathological stage in RCC and with tumor grade in BCA patients. CONCLUSION Circulating mtDNA levels are associated with gender and age in healthy individuals, but not in cancer patients. Quantification of circulating mtDNA may help identify patients with urologic malignancies.

The use of a colon cancer associated nuclear antigen CCSA-2 for the blood based detection of colon cancer†

The early diagnosis of colorectal cancer (CRC) is central for effective treatment, as prognosis is directly related to the stage of the disease. Development of tumor markers found in the blood from patients, which can detect CRC at an early stage, should have a major impact in morbidity and mortality of this disease. The nuclear matrix is the structural scaffolding of the nucleus and specific nuclear matrix proteins (NMPs) have been identified as an “fingerprint” for various cancer types. Previous studies from our laboratory have identified four colon cancer associated NMPs termed colon cancer‐specific antigen (CCSA)‐2 to (CCSA)‐5. The objective of the present study was to analyze the expression of one of these proteins, CCSA‐2 in serum from various patient populations and to determine whether CCSA‐2 antibodies could be used in a clinically applicable serum‐based immunoassay specifically to detect colon cancer. Using an indirect ELISA, which detects CCSA‐2, the protein was measured in the serum from 174 individuals, including healthy individuals, patients with colon cancer, patients with diverticulosis, colon polyps, inflammatory bowel disease (IBD) as well as other cancer types. With a predetermined cutoff absorbance of 0.6 OD we have successfully utilized this approach to develop an immunoassay that detected colon cancer. The immunoassay showed a sensitivity of 88.8% (24/27) and an overall specificity of 84.2% (106/127). This initial study showed the potential of CCSA‐2 to serve as a highly specific blood based marker for colon cancer. Although potentially promising, the results of this study must be confirmed in larger independent validation studies. J. Cell. Biochem. 104: 286–294, 2008.

An anomalous muscle predisposing to the development of hypothenar hammer syndrome

The so-called hypothenar hammer syndrome is a rare entity caused by occlusion of the ulnar artery secondary to repetitive trauma to the hypothenar eminence and is typically found in persons working with vibrating tools. Its clinical symptoms are pain, cold sensitivity, numbness, weakness or stiffness, and discoloring of the affected fingers. The repetitive trauma most likely disrupts the internal elastic lamina, which can lead to complete occlusion of the ulnar artery within the Guyon canal. Anatomical variations may promote the onset of a thrombosis. Typical clinical signs following occlusion are absent flow (as seen in the Allen test) and decreased sensibility in the affected area and decreased capillary refill.1,2

Non-invasive proteomics—thinking about personalized breast cancer screening and treatment

The early diagnosis of breast cancer in potentially curable stages improves prognosis and consecutively reduces mortality of breast cancer patients. Established screening programs have an unfavorable connotation due to significant rates of false negative as well as false positive results leading to overdiagnosis and overtherapy. The combination of a non-invasive breast-cancer-suspectability-biomarker with established clinical diagnostics could help to increase the acceptance of population based breast cancer screening programs by creating an individual risk profile, which is irrespective of mammography quality and interpretation. Recently, non-invasive proteomic biomarkers obtained from blood, saliva or nipple aspiration fluid have been extensively investigated and might play a future role in the personalized management of breast cancer screening. A simple, robust and inexpensive, non-invasive test for screening and diagnosis could easily be performed in every medical practice leading to an affordable, high-throughput instrument. This review describes recently investigated proteomic screening biomarkers that could improve the early diagnosis of breast cancer in the following years.

Increased Detection of Lymphatic Vessel Invasion by D2-40 (Podoplanin) in Early Breast Cancer: Possible Influence on Patient Selection for Accelerated Partial Breast Irradiation

PURPOSE Several international trials are currently investigating accelerated partial breast irradiation (APBI) for patients with early-stage breast cancer. According to existing guidelines, patients with lymphatic vessel invasion (LVI) do not qualify for APBI. D2-40 (podoplanin) significantly increases the frequency of LVI detection compared with conventional hematoxylin and eosin (HE) staining in early-stage breast cancer. Our purpose was to retrospectively assess the hypothetical change in management from APBI to whole breast radiotherapy with the application of D2-40. PATIENTS AND METHODS Immunostaining with D2-40 was performed on 254 invasive breast tumors of 247 patients. The following criteria were used to determine the eligibility for APBI: invasive ductal adenocarcinoma of < or =3 cm, negative axillary node status (N0), and unifocal disease. Of the 247 patients, 74 with available information concerning LVI, as detected by D2-40 immunostaining and routine HE staining, formed our study population. RESULTS Using D2-40, our results demonstrated a significantly greater detection rate (p = .031) of LVI compared with routine HE staining. LVI was correctly identified by D2-40 (D2-40-positive LVI) in 10 (13.5%) of 74 tumors. On routine HE staining, 4 tumors (5.4%) were classified as HE-positive LVI. Doublestaining of these specimens with D2-40 unmasked false-positive LVI status in 2 (50%) of the 4 tumors. According to the current recommendations for APBI, immunostaining with D2-40 would have changed the clinical management from APBI to whole breast radiotherapy in 8 (10.8%) of 74 patients and from whole breast radiotherapy to APBI in 2 patients (2.7%). CONCLUSION These data support the implementation of D2-40 immunostaining in the routine workup to determine a patients eligibility for APBI.

METHOD COMPARISON FOR CA 15-3, CA 19-9, AND CA 125 DETERMINATION USING THE NEW LOCI TECHNIQUE OF DIMENSION VISTA 1500 AND IMMULITE 2000 XPI

We performed method comparison for the tumor markers CA 15-3, CA 19-9, and CA 125 measured by luminescent oxygen channeling immunoassay technology on the Dimension Vista 1500 and by classic luminescence technology on the Immulite 2000 XPI. Within-day and total imprecision were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines using three serum pools at different clinically relevant levels. In addition, parallel measurements on both systems were performed in a total of 738 routine serum samples (133 CA 15-3, 395 CA 19-9, and 210 CA 125). Total imprecision of serum pools for CA 15-3 ranged between 4.6% and 5.9%, for CA 19-9 between 4.4% and 7.8%, and for CA 125 between 3.3% and 4.3%. Marker values determined within the measurement range of both systems correlated well with each other (R = 0.88 for CA 15-3, R = 0.93 for CA 19-9, and R = 0.96 for CA 125). Slopes between the Vista and the Immulite method were 0.96 for CA 125, 0.72 for CA 15-3, and 0.87 for CA 19-9, indicating lower values for CA 15-3 and CA 19-9 when measured by the Vista method. This was particularly obvious for CA 19-9 levels in the lower measuring range of <100 U/mL (R = 0.85; slope 0.73).

Identification of specific nuclear structural protein alterations in human breast cancer

Breast cancer is the most commonly diagnosed type of cancer and a major cause of death in women. Reliable biomarkers are urgently needed to improve early detection or to provide evidence of the prognosis for each individual patient through expression levels in tumor tissue or body fluids. This proteomic analysis focused on the nuclear structure of human breast cancer tissue, which has been shown to be a promising tool for cancer biomarker development. The nuclear matrix composition of human breast cancer (n = 14), benign controls (n = 2), and healthy controls (n = 2) was analyzed by high‐resolution two‐dimensional gel electrophoresis and mass spectrometry. Validation studies were performed in an individual sample set consisting of additional breast cancer tissues (n = 3) and additional healthy control tissues (n = 2) by one‐dimensional immunoblot. In this setting, we identified five proteins that were upregulated in human breast cancer tissue, but absent in the healthy and benign controls (P < 0.001). These spots were also present in the investigated human breast cancer cell lines, but absent in the MCF10a cell line, which represents normal human epithelial breast cells. Two of the breast cancer‐specific proteins have been confirmed to be calponin h2 and calmodulin‐like protein 5 by one‐dimensional immunoblot. This is the first study demonstrating the expression of both proteins in human breast cancer tissue. Further studies are required to investigate the potential role of these proteins as biomarkers for early diagnosis or prognosis in human breast cancer. J. Cell. Biochem. 112: 3176–3184, 2011.

Surgical Strategies in the Treatment of Gynecomastia Grade I-II: The Combination of Liposuction and Subcutaneous Mastectomy Provides Excellent Patient Outcome and Satisfaction

Background: Gynecomastia (GM) is a benign condition with glandular tissue enlargement of the male breast. GM is classified into 4 grades of increasing severity. We describe a series of GM grade I-II, diagnosed, treated surgically and analyzed regarding feasibility, complication rate, and satisfaction. Methods: From 2005 to 2012, a chart review was performed for 53 patients. Preoperative examination included endocrine and urological examination and exclusion of other pathological conditions. The surgical technique consisted of liposuction through an inframammarian-fold incision and excision of the glandular tissue by a minimal periareolar approach. Results: A total number of 53 male patients with 104 breasts were available for analysis. By liposuction, a median of 300 ml (range: 10-1000 ml) was aspirated from each breast and 25.1 g (range: 3-233 g) gland tissue was resected. Surgery lasted between 25 and 164 min per patient (median: 72 min). 2 postoperative hemorrhages occurred (n = 2, 3.8%). 2 patients underwent re-operation due to cosmetic reasons (n = 2, 3.8%). Conclusions: This analysis demonstrates that treatment of GM grade I-II can easily be performed by liposuction combined with subcutaneous resection of the glandular tissue as a minimally invasive and low-impact surgical treatment with a low rate of complications and excellent patient satisfaction. Preoperative workup is important to rule out specific diseases or malignancy causing the GM.