Distinct Expression and Function of FcεRII in Human B Cells and Monocytes.

FcεRII is a multifunctional low-affinity IgER that is involved in the pathogenesis of allergic, inflammatory, and neoplastic diseases. Although discrepancies in FcεRII-mediated functions are being increasingly recognized, the consequences of FcεRII activation are not completely understood. In this study, we evaluated the expression of FcεRII on human blood cells and found that it was primarily expressed on monocytes and B cells. Although IL-4 promoted expression of the FcεRIIb isoform on B cells and monocytes, the expression of the FcεRIIa isoform was not dependent on IL-4. Furthermore, FcεRII predominantly bound allergen-IgE complexes on B cells but not on monocytes. FcεRII-mediated allergen-IgE complex uptake by B cells directed Ags to MHC class II-rich compartments. FcεRII-bearing monocytes and B cells expressed high levels of the FcεRII sheddase a disintegrin and metalloproteinase 10, which implies that they are important sources of soluble FcεRII. Moreover, we identified that IgE immune complex stimulation of FcεRII activated intracellular tyrosine phosphorylation via Syk in B cells but not in monocytes. Importantly, FcεRII-mediated signaling by allergen-IgE immune complexes increased IFN-γ production in B cells of allergic patients during the build-up phase of allergen-specific immunotherapy. Together, our results demonstrate that FcεRII mediates cell type-dependent function in allergic reactions. In addition, the results identify a novel allergen-IgE complex/FcεRII/Syk/IFN-γ pathway in allergic responses and suggest that FcεRII may play a role in regulating allergic reactions via modulating IFN-γ production in B cells.

Management of Capsular Contracture in Cases of Silicone Gel Breast Implant Rupture with Use of Pulse Lavage and Open Capsulotomy.

INTRODUCTION: Pulse lavage (PL) irrigation of prosthesis pockets has prior been described for breast implant salvages. However, PL for removal of leaked silicone from prosthesis pockets after implant ruptures has not been studied yet. Since open capsulotomies are regarded as equal treatment of capsular contracture (CC) than capsulectomies, this study analyzed the clinical outcome of PL for silicone removal and subsequent capsulotomy in cases of concurrent CC and breast implant rupture. METHODS: Between 2012 and 2017, 55 patients (75 breasts) with suspected silicone implant rupture and CC (Baker grade III/IV), after primary breast augmentation or implant-based breast reconstruction, were included in a retrospective, observational study. Mean patient follow-up was 12.2 ± 3.6 months. RESULTS: In all preoperatively suspected ruptured silicone breast implants, around a quarter were intact. In contrast to previously published data, implant exchanges in cases of implant ruptures did not lead to significantly higher CC recurrence rates (27.6% vs. 22.2% in cases of intact implants, p = 0.682), if the prosthesis pockets were treated with PL irrigation followed by open capsulotomy. PL reduced the amount of encapsulated silicone remnants histologically. The age of patients with CC after failed implant-based reconstruction was significant lower for salvage surgeries with flap reconstruction than for implant exchanges, p < 0.05. CONCLUSIONS: PL irrigation of prosthesis pockets prior to open capsulotomy is a safe and effective treatment of CC with concurrent silicone leakage. Remaining silicone remnants in breast capsules may affect the development of a recurrent CC. To avoid CC recurrences, patients should consider conversion to autologous tissue. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Calponin-h2: a potential serum marker for the early detection of human breast cancer?

Early diagnosis is the key for the successful treatment of breast cancer. A serum marker for the early detection of breast cancer could significantly reduce breast cancer morbidity and mortality by bringing the time of diagnosis at an earlier and therefore still curable stage. So far, no biomarker for the early detection is available for the clinical routine. The aim of the present study was to evaluate the use of calponin-h2 as a blood-based biomarker for the early diagnosis of this disease. Using two monoclonal antibodies against calponin-h2, we developed a sandwich ELISA to analyze the serum levels of calponin-h2. In order to evaluate the diagnostic potential of this biomarker, patients with breast cancer (n = 76), benign diseases of the breast (n = 51) and healthy females (n = 24) were analyzed. Serum levels above 10 ng/ml were only observed in patients with breast cancer (n = 8; 10.5%). Further large-scale studies and preanalytic evaluations are necessary to clarify the definite role of calponin-h2 as a biomarker in breast cancer management.

Method comparison for determination of the tumor markers AFP, CEA, PSA and free PSA between Immulite 2000 XPI and Dimension Vista 1500.

BACKGROUND: For the Luminescent Oxygen Channeling Immunoassay (LOCI) technology as established for Dimension Vista 1500, assays have been developed for the serum tumor markers AFP, CEA, PSA and free PSA. We performed a method analysis for these parameters using the Immulite 2000 XPI. METHODS: Determination of within-day and total imprecision of the methods was carried out according to CLSI guidelines with three serum pools. In addition, parallel measurements were performed with both systems in 1,871 routine serum samples and correlations were calculated. RESULTS: Calculated total imprecision of the three serum pools for AFP was 3.8 - 4.3%, for CEA 3.3 - 4.3%, for tPSA 3.6 - 4.0% and for fPSA it was 3.5 - 8.2%. Correlations of these markers across the entire value range were very good with the following correlation coefficients: 0.997 for AFP, 0.996 for CEA, 0.971 for tPSA and 0.988 for fPSA. While values for AFP and tPSA from both methods were comparable (slopes 1.02 and 1.01), lower values were measured for CEA and fPSA with the Dimension Vista (slopes 0.83 and 0.91). For AFP, a sample cluster with considerably higher values than with Dimension Vista was observed in the lower measurement range (< 20 ng/mL). CONCLUSIONS: The assays for AFP, CEA, tPSA and fPSA, as developed with the LOCI technology for the Dimension Vista, show good comparability with results obtained from the Immulite 2000 XPI. However, lower measurement ranges for CEA and fPSA as well as individual divergences, especially with AFP, must be taken into consideration in the event of method changeover.

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).

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. METHODS: Serum from patients with prostate cancer (n = 24), bladder cancer (n = 24), renal cell carcinoma (n = 24) and control subjects (n = 48) was spiked with cel-miR-39, and then ribonucleic acid was isolated. Quantitative real-time polymerase chain reaction was used to determine the levels of candidate reference genes (RNU1-4, RNU6-2, SNORD43, SNORD44, SNORD48, SNORA74A, miR-let-7a-1, miR-106a). Reference gene stability was determined using the NormFinder, geNorm and comparative delta-Ct algorithm. The effect of normalization was tested with miR-21 as the target gene, as this was previously suggested to be upregulated in cancer patients' serum. RESULTS: Recovery of cel-miR-39 (mean 11.6%, range 1-56%) was similar in control subjects and cancer patients. SNORD44 and SNORD74A levels were around the detection limit of the assay and were thus omitted. All remaining candidates showed satisfying stability; SNORD43 was the most stable reference gene using all three algorithms. A combination of two genes (SNORD43, RNU1-4) increases the stability somewhat. The level of miR-21 was similar in cancer patients and healthy controls, irrespective of the normalization strategy. CONCLUSIONS: SNORD43 is a suitable reference gene for the analysis of circulating micro-ribonucleic acid in patients with urological malignancies. Our study questions the suitability of miR-21 as a biomarker for uro-oncological patients.

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.

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.

Clinical Performance of CEA, CA19-9, CA15-3, CA125 and AFP in Gastrointestinal Cancer Using LOCI™-based Assays.

BACKGROUND/AIM: Few data are available regarding the clinical performance of LOCI™-based tumor marker assays. We investigated the diagnostic power of carcinogenic antigen, carbohydrate antigen 19-9, carbohydrate antigen 15-3, carbohydrate antigen 125 and alpha-fetoprotein for detection of gastrointestinal (GI) cancer. PATIENTS AND METHODS: We analyzed sera from 204 patients (107 with GI cancer, 73 with benign GI diseases and 24 healthy controls) using the Dimension™ Vista1500 analyzer. RESULTS: Levels of biomarkers in healthy controls were in the expected ranges and were only slightly higher in benign GI controls. Established tumor-type-associated markers were elevated in specific cancer types and discriminated well between cancer and benign controls. Best performance was found for CEA in colorectal cancer (area under the curve=0.84, sensitivity=51.7% at 95% specificity vs. benign), CA19-9 in gallbladder/pancreatic cancer (AUC=0.85, sensitivity=60.6%) and AFP in liver cancer (AUC=0.87, sensitivity=68.4%). CONCLUSION: Our study demonstrated the high diagnostic power of well-known biomarkers. LOCI™-based tumor marker assays give reliable results in routine diagnostics.

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.

Specific expression of k63-linked ubiquitination of calmodulin-like protein 5 in breast cancer of premenopausal patients.

PURPOSE: Posttranslational modifications such as ubiquitination regulate many functions of proteins by affecting their interaction with other molecules, their activity, and their subcellular localization. In cancer biology, the ubiquitin network has gained major interest. K63-linked ubiquitination has emerged as a posttranslational modification with functional consequences, as it acts in several processes such as protein trafficking, DNA repair, and inflammation. Moreover, k63-linked ubiquitination is involved in the regulation of carcinogenesis. Based on previous findings, the aim of this study was to evaluate the ubiquitination of CALML5 in breast cancer patients. PATIENTS AND METHODS: The breast cancer cell lines SkBr3, MCF7, HCC1937, and BT474 as well as 23 tumor samples of patients with primary breast cancer and the normal adjacent breast tissue were analyzed by one-dimensional immunoblot. RESULTS: Using specific antibodies against CALML5 and k63-linked ubiquitin, we demonstrate a k63-linked ubiquitination in the nuclear fraction of premenopausal breast cancer patients. K63-linked ubiquitination of CALML5 was found in breast cancer tissue, but not found in surrounding healthy tissue. CONCLUSION: Our findings support the concept that ubiquitination of CALML5 in the nucleus is involved in the carcinogenesis of breast cancer in premenopausal women.

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.

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.

A Rare Complication Following Breast Implant Surgery: Capsular Contracture with a Cutaneous Silicone Fistula after Breast Reconstruction with Silicone Gel Implants.

SUMMARY: BACKGROUND: We report the case of a 74-year-old female patient who presented at the Breast Care Centre with watery discharge from a fistula in the inframammary fold of the left breast. CASE REPORT: The patient initially presented with watery discharge coming from the fistula, which later took on a more viscous consistency. She reported mild discomfort as well as mild erythema. Clinical examination and diagnostic approaches including magnetic resonance imaging (MRI) revealed a cutaneous silicone fistula as a rare complication following breast implant reconstruction. The condition was treated with excision of the fistula and bilateral implant removal. CONCLUSIONS: This case report documents a rare complication following breast reconstruction with implants, and is to our knowledge the first described MRI-detected cutaneous silicone fistula.

MicroRNAs in renal cell carcinoma: diagnostic implications of serum miR-1233 levels.

BACKGROUND: MicroRNA expression is altered in cancer cells, and microRNAs could serve as diagnostic/prognostic biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC). METHODOLOGY/PRINCIPAL FINDINGS: We first explored microrna expression profiles in tissue and serum using taqman low density arrays in each six malignant and benign samples: Although 109 microRNAs were circulating at higher levels in cancer patients' serum, we identified only 36 microRNAs with up-regulation in RCC tissue and serum of RCC patients. Seven candidate microRNAs were selected for verification based on the finding of up-regulation in serum and tissue of RCC patients: miR-7-1*, miR-93, miR-106b*, miR-210, miR-320b, miR-1233 and miR-1290 levels in serum of healthy controls (n = 30) and RCC (n = 33) patients were determined using quantitative real-time PCR (TaqMan MicroRNA Assays). miR-1233 was increased in RCC patients, and thus validated in a multicentre cohort of 84 RCC patients and 93 healthy controls using quantitative real-time PCR (sensitivity 77.4%, specificity 37.6%, AUC 0.588). We also studied 13 samples of patients with angiomyolipoma or oncocytoma, whose serum miR-1233 levels were similar to RCC patients. Circulating microRNAs were not correlated with clinical-pathological parameters. CONCLUSIONS/SIGNIFICANCE: MicroRNA levels are distinctly increased in cancer patients, although only a small subset of circulating microRNAs has a tumor-specific origin. We identify circulating miR-1233 as a potential biomarker for RCC patients. Larger-scaled studies are warranted to fully explore the role of circulating microRNAs in RCC.

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 patient's eligibility for APBI.