Comparison of different label-free imaging high-throughput biosensing systems for aptamer binding measurements using thrombin aptamers.

To enable the analysis of several hundreds to thousands of interactions in parallel, high-throughput systems were developed. We used established thrombin aptamer assays to compare three such high-throughput imaging systems as well as analysis software and user influence. In addition to our own iRIf-system, we applied bscreen and IBIS-MX96. As non-imaging reference systems we used Octet-RED96, Biacore3000, and Monolith-NT.115. In this study we measured 1378 data points. Our results show that all systems are suitable for analyzing binding kinetics, but the kinetic constants as well as the ranking of the selected aptamers depend significantly on the applied system and user. We provide an insight into the signal generation principles, the systems and the results generated for thrombin aptamers. It should contribute to the awareness that binding constants cannot be determined as easily as other constants. Since many parameters like surface chemistry, biosensor type and buffer composition may change binding behavior, the experimenter should be aware that a system and assay dependent KD is determined. Frequently, certain conditions that are best suited for a given biosensing system cannot be transferred to other systems. Therefore, we strongly recommend using at least two different systems in parallel to achieve meaningful results.

De-Escalation Strategies in Human Epidermal Growth Factor Receptor 2 (HER2)-Positive Early Breast Cancer (BC): Final Analysis of the West German Study Group Adjuvant Dynamic Marker-Adjusted Personalized Therapy Trial Optimizing Risk Assessment and Therapy Response Prediction in Early BC HER2- and Hormone Receptor-Positive Phase II Randomized Trial-Efficacy, Safety, and Predictive Markers for 12 Weeks of Neoadjuvant Trastuzumab Emtansine With or Without Endocrine Therapy (ET) Versus Trastuzumab Plus ET.

Purpose Human epidermal growth factor receptor 2 (HER2)-positive/hormone receptor (HR)-positive breast cancer is a distinct subgroup associated with lower chemotherapy sensitivity and slightly better outcome than HER2-positive/HR-negative disease. Little is known about the efficacy of the combination of endocrine therapy (ET) with trastuzumab or with the potent antibody-cytotoxic, anti-HER2 compound trastuzumab emtansine (T-DM1) with or without ET for this subgroup. The West German Study Group trial, ADAPT (Adjuvant Dynamic Marker-Adjusted Personalized Therapy Trial Optimizing Risk Assessment and Therapy Response Prediction in Early Breast Cancer) compares pathologic complete response (pCR) rates of T-DM1 versus trastuzumab with ET in early HER2-positive/HR-positive breast cancer. Patients and Methods In this prospective, neoadjuvant, phase II trial, 375 patients with early breast cancer with HER2-positive and HR-positive status (n = 463 screened) were randomly assigned to 12 weeks of T-DM1 with or without ET or to trastuzumab with ET. The primary end point was pCR (ypT0/is/ypN0). Early response was assessed in 3-week post-therapeutic core biopsies (proliferation decrease ≥ 30% Ki-67 or cellularity response). Secondary end points included safety and predictive impact of early response on pCR. Adjuvant therapy followed national standards. Results Baseline characteristics were well balanced among the arms. More than 90% of patients completed the therapy per protocol. pCR was observed in 41.0% of patients treated with T-DM1, 41.5% of patients treated with T-DM1 and ET, and 15.1% with trastuzumab and ET ( P < .001). Early responders (67% of patients with assessable response) achieved pCR in 35.7% compared with 19.8% in nonresponders (odds ratio, 2.2; 95% CI, 1.24 to 4.19). T-DM1 was associated with a significantly higher prevalence of grade 1 to 2 toxicities, especially thrombocytopenia, nausea, and elevation of liver enzymes. Overall toxicity was low; seventeen therapy-related severe adverse events (T-DM1 arms v trastuzumab plus ET; 5.3% v 3.1%, respectively) were reported. Conclusion The ADAPT HER2-positive/HR-positive trial demonstrates that neoadjuvant T-DM1 (with or without ET) given for only 12 weeks results in a clinically meaningful pCR rate. Thus, a substantial number of patients are spared the adverse effects of systemic chemotherapy.

West German Study Group Phase III PlanB Trial: First Prospective Outcome Data for the 21-Gene Recurrence Score Assay and Concordance of Prognostic Markers by Central and Local Pathology Assessment.

PURPOSE: The 21-gene Recurrence Score (RS) assay is a validated prognostic/predictive tool in early hormone receptor-positive breast cancer (BC); however, only a few prospective outcome results have been available so far. In the phase III PlanB trial, RS was prospectively used to define a subset of patients who received only endocrine therapy. We present 3-year outcome data and concordance analysis (among biomarkers/RS). PATIENTS AND METHODS: Central tumor bank was established prospectively from PlanB (intermediate and high-risk, locally human epidermal growth factor receptor 2-negative BC). After an early amendment, HR-positive, pN0-1 patients with RS ≤ 11 were recommended to omit chemotherapy. RESULTS: From 2009 to 2011, PlanB enrolled 3,198 patients with a median age of 56 years; 41.1% had node-positive and 32.5% grade 3 disease. In 348 patients (15.3%), chemotherapy was omitted based on RS ≤ 11. After 35 months median follow-up, 3-year disease-free survival in patients with RS ≤ 11 and endocrine therapy alone was 98% versus 92% and 98% in RS > 25 and RS 12 to 25 in chemotherapy-treated patients, respectively. Nodal status, central and local grade, the Ki-67 protein encoded by the MKI67 gene, estrogen receptor, progesterone receptor, tumor size, and RS were univariate prognostic factors for disease-free survival; only nodal status, both central and local grade, and RS were independent multivariate factors. Histologic grade was discordant between central and local laboratories in 44%. RS was positively but moderately correlated with the Ki-67 protein encoded by the MKI67 gene and grade and negatively correlated with progesterone receptor and estrogen receptor. CONCLUSION: In this prospective trial, patients with enhanced clinical risk and omitted chemotherapy on the basis of RS ≤ 11 had excellent 3-year survival. The substantial discordance observed between traditional prognostic markers and RS emphasizes the need for standardized assessment and supports the potential integration of standardized, well-validated genomic assays such as RS with clinicopathologic prognostic factors for chemotherapy indication in early hormone receptor-positive BC.

Breast conservative surgery and local recurrence.

INTRODUCTION: Breast conservation is a legacy of Umberto Veronesi who laid the groundwork for the preservation of the body image of women affected by breast cancer (BC) with the Milan I study in the late 70ies of the last millennium. Breast conservative surgery (BCS) has two aspects: oncological safety of tumour resection with free margins and aesthetic preservation of the breast. Determinants of local control used to be T-size, nodal status and receptor status until biologically driven concepts defined risk of recurrence on the basis of molecular portraits. We explored whether these concepts of intrinsic subtypes prove at a large scale in the context of BCS and which surgical techniques procure best oncological and aesthetic outcomes, avoiding re-excision and necessity of conversion to mastectomy. PATIENTS AND METHODS: We analyzed 1035 BCS patients with primary unilateral breast cancer (2004-2009) with regards to the local recurrence as a function of tumour location, surgical technique, resection volume, T-size, nodal status, grading, histopathological and intrinsic subtype and margins. RESULTS: Five surgical techniques were applied to 944 eligible patients at a median follow-up of 5.2 years with the following frequency: Glandular rotation mammoplasty (63.8%), tumour-adapted rotation mammoplasty (20.9%), dermoglandular rotation mammoplasty (6.7%), 4.4% (lateral thoracic wall advancement), 0.7% latissimus dorsi flap (0.7%) and others (13.5%). Tumour-free margins were achieved in 88.6% of all patients at first surgery. Recurrence was independent of the surgical technique used, resection volume, T-size (in a T1/T2-cohort), nodal status (in low N-stages: NO/N1) and histopathology (inv.-ductal vs. lobular), however non-invasive subtype (DCIS), high grading (G3 vs. G1), non-luminal Her2 positive BC and triple-negative breast cancer (TNBC) were significantly associated with local recurrence. CONCLUSIONS: Five defined oncoplastic principles presented in our nomogramme (targeted breast surgery) allow the reconstruction of major segmental resection defects during breast-conserving therapy with high clinical applicability and result in favorable oncological and aesthetic outcome. Recurrence was not a function of traditional prognostic factors like T-size or nodal status (in a T1/T2, N0/N1 cohort), but of grading, intrinsic subtypes and non-invasive breast cancer components. Lobular histology, multi-centricity and DCIS were predictive for breast preservation failure and conversion to mastectomy.

Intraoperative Boost Radiotherapy during Targeted Oncoplastic Breast Surgery: Overview and Single Center Experiences.

Breast-conserving surgery followed by whole-breast irradiation is the standard local therapy for early breast cancer. The international discussion of reduced importance of wider tumor-free resection margins than "tumor not touching ink" leads to the development of five principles in targeted oncoplastic breast surgery. IORT improves local recurrence risk and diminishes toxicity since there is less irradiation of healthy tissue. Intraoperative radiotherapy (IORT) can be delivered in two settings: an IORT boost followed by a conventional regimen of external beam radiotherapy or a single IORT dose. The data from TARGIT-A and ELIOT reinforce the conviction that intraoperative radiotherapy during breast-conserving surgery is a reliable alternative to conventional postoperative fractionated irradiation, but only in a carefully selected population at low risk of local recurrence. We describe our experiences with IORT boost (50 kV energy X-rays; 20 Gy) in combination with targeted oncoplastic breast surgery in a routine clinical setting. Our experiences demonstrate the applicability and reliability of combining IORT boost with targeted oncoplastic breast surgery in breast-conserving therapy of early breast cancer.

Filariasis of the axilla in a patient returning from travel abroad: a case report.

BACKGROUND: The term filariasis comprises a group of parasitic infections caused by helminths belonging to different genera in the superfamily Filaroidea. The human parasites occur mainly in tropical and subtropical regions, but filariae are also found in temperate climates, where they can infect wild and domestic animals. Humans are rarely infected by these zoonotic parasites. PATIENTS AND METHODS: A 55-year-old patient presented with a new-onset, subcutaneous, non-tender palpable mass in the right axilla. Ultrasonography showed a 1.3-cm, solid, singular encapsulated node. Sonography of the breast on both sides, axilla and lymphatic drainage on the left side, lymphatic drainage on the right side, and mammography on both sides were without pathological findings. The node was excised under local anesthesia as the patient refused minimal invasive biopsy. RESULTS: On histopathological examination, the tail of a parasite of the group of filariae was found. The patient revealed that she had stayed in Africa and Malaysia for professional reasons. 6 months before the time of diagnosis, she had also suffered from a fever and poor general condition after a trip abroad. The patient was referred for further treatment to the Institute for Tropical Medicine at the University of Dusseldorf, where a treatment with ivermectin was conducted on the basis of positive staining with antibodies against filariae. CONCLUSION: Our case demonstrates the importance of interdisciplinary collaboration between breast center, pathology, and other specialties such as microbiology and tropical medicine.

Functional lumen imaging probe to assess geometric changes in the esophagogastric junction following endolumenal fundoplication.

BACKGROUND: The functional lumen imaging probe (FLIP) uses impedance planimetry to measure the geometry of a distensible organ. The purpose of this study was to evaluate FLIP as a method to determine structural changes at the gastroesophageal junction (GEJ) following transoral incisionless fundoplication (TIF) and compare these findings with the accepted methods of esophageal testing. METHODS: Two different approaches (TIF1.0 and 2.0) using the EsophyX™ device were performed in six and five animals, respectively. Three dogs underwent a sham procedure. FLIP measurements were performed pre- and post-procedure and at 2-week follow-up. Upper endoscopy, manometry, and 48-h pH testing were also performed at each time point. FLIP was performed in ten patients before and 3 months after TIF. RESULTS: Following TIF procedures, there was a significant decrease in cross-sectional area (CSA) of GEJ compared to baseline; however, the CSA of both groups returned to baseline at 2-week follow-up. The FLIP results were supported with pH testing and correlated highly with both measures of GEJ structural integrity (LES and cardia circumference). Following TIF in humans, there was a decrease in GEJ distensibility compared to baseline that persisted to the 3-month evaluation. CONCLUSION: FLIP is able to measure and display changes in tissue distensibility at the GEJ, and results correlate with established methods of testing. FLIP may represent a single testing modality by which to diagnose GERD and evaluate the outcome after antireflux surgery.

The Hill antireflux repair at 5 institutions over 25 years.

BACKGROUND: Long-term (> 5 years) studies of antireflux operations are needed. This study evaluates long-term results of the open Hill repair at multiple institutions. METHODS: This is a retrospective cohort study of open Hill repairs from 1972 to 1997 at 5 North American medical centers with a mean follow-up of 10 years. Objective data and standardized clinical outcomes were collected at a central site. Subjective results, medication use, and satisfaction scales were obtained through scripted phone interview. Results between 2 Hill-trained centers and 3 independent centers were compared. RESULTS: One thousand one hundred eighty-one patients met the inclusion criteria. Symptomatic improvement was found in 97% and good to excellent results in 93%. Medication use was markedly reduced. Hiatal hernia recurrence was found in 77 (6.9%); the reoperation rate was 1.9%. Differences in outcomes between Hill centers and independent centers were minor. CONCLUSIONS: Excellent results with the open Hill repair are durable beyond 10 years and are reproducible. Anatomic recurrence and reoperative rates are low.

Three-Tesla magnetic resonance imaging of the wrist: diagnostic performance compared to 1.5-T.

PURPOSE: To assess the diagnostic capability of a new multi-contrast high-resolution imaging protocol for the wrist at 3 T and to compare it to our standard 1.5-T protocol. MATERIALS AND METHODS: In 10 volunteers, images were acquired at 1.5 T (standard surface coil) and 3 T (custom-made phased array coil; Gyroscan Intera; Philips Medical Systems, Best, the Netherlands). Imaging protocols consisted of coronal T1-weighted turbo spin-echo (TSE), coronal T2-weighted TSE, and proton density-weighted TSE fat-saturated (FS) sequences and sagittal T2-TSE and proton density-weighted TSE-FS sequences. Increased signal-to-noise ratio at 3 T was used to reduce measured voxel size from 0.50 x 0.50 x 3.0 mm (1.5 T) to 0.20 x 0.20 x 1.5 mm (3 T). Total examination time was approximately 25 minutes. To compare the diagnostic capability of both protocols, 4 observers assessed in consensus the visibility of 14 well-defined clinically important anatomical landmarks (origin and insertion of 6 intrinsic and extrinsic carpal ligaments, central portion of the triangular fibrocartilage complex, and ulna, triquetral, and radial attachments). Image quality and artifacts were ranked qualitatively on a 5-point scale. RESULTS: Of 140 detectable structures, 75 were detected at 1.5 T and 126 at 3 T. Overall image quality was significantly better at 3 T (3.8 vs 4.9, P = 0.002), whereas artifact score did not differ significantly (4.3 vs 4.5, P = 0.317). CONCLUSIONS: This is the result of investing the higher signal-to-noise ratio at 3 T into better spatial resolution; depiction of the normal anatomy of the wrist benefits significantly. Additional studies will need to be performed to determine if 3-T images will increase the accuracy of detecting abnormalities of these structures of the wrist.

Transoral endoscopic fundoplication in the treatment of gastroesophageal reflux disease: the anatomic and physiologic basis for reconstruction of the esophagogastric junction using a novel device.

OBJECTIVE: To determine the safety, mechanism of action, immediate postprocedural anatomic impact on the esophagogastric junction, and short-term efficacy of the first entirely endolumenal antireflux procedure. BACKGROUND: A safe and effective endoscopic antireflux procedure remains elusive. Transoral endolumenal surgery has enormous potential for the treatment of gastroesophageal reflux disease (GERD) and other esophagogastric diseases. A canine model was used to study a novel endoscopic device, which allows for creation of an endoluminal fundoplication. METHODS: The transoral incisionless fundoplication (TIF) was performed in 21 canines in a phase I feasibility and safety study, and in 21 canines in a phase II study that included a detailed objective assessment of the effects of 2 variants of the TIF procedure (TIF 1.0 and TIF 2.0) versus sham on esophageal physiology and esophagogastric junction (EGJ) anatomy. RESULTS: In phase I, TIF provided a safe and feasible endolumenal therapy for GERD, with histologic data that demonstrated serosal fusion of approximated full-thickness tissue plications and durability of the fundoplication. TIF procedures effectively reduced cardia circumference and improved Hill classification grade. In phase II, the TIF 2.0 procedure achieved normalization of distal esophageal acid exposure and increased lower esophageal sphincter (LES) pressure and length based on objective testing over a 2-week period. TIF 2.0 demonstrated superior results to TIF 1.0, and valve appearance and location exhibited similarity to the Nissen fundoplication by vector volume analysis. CONCLUSIONS: The TIF procedure is safe and results in a durable and functional fundoplication as well as a platform for further development and modification of the procedure, which can be use to impact outcome. This work provides the foundation for human translation and assessment of long-term outcomes.

Neointimal expression of rapamycin receptor FK506-binding protein FKBP12: postinjury animal and human in-stent restenosis tissue characteristics.

Despite excellent clinical results for sirolimus (rapamycin)-eluting stents, the exact mechanisms of antirestenotic activity and affected cellular targets are incompletely understood. Therefore, we determined the presence and tem- porospatial expression pattern of FKBP12, the primary intracellular receptor of rapamycin, in rat carotid arteries after balloon injury, as well as in human in-stent restenosis and primary stable coronary atheroma. FKBP12 expression was assessed by immunohistochemistry. Rat carotid arteries revealed maximal expression in 57.7 +/- 4.0% of neointimal cells at day 7. A large proportion of these FKBP12+ cells showed luminally confined co-expression with dendritic cell markers. Despite a considerably thicker neointima at day 28, presence of FKBP12 decreased (8.5 +/- 1.9%, p = 0.02) with a scattered pattern in luminal and deep neointima. Likewise, human in-stent restenosis atherectomy specimens (time after stent implantation 2-12 months) revealed a comparable extent of cellular rapamycin receptor expression (9.3 +/- 1.0%) that significantly differed from that found in primary stable atheroma (1.3 +/- 0.4%, p < 0.001). In conclusion, the rapamycin receptor is predominantly present during early neointima formation, while mature neointimal atheromas show a relatively low expression without confinement to luminal areas. Co-expression of FKBP12 and dendritic cell markers suggests that dendritic cells may be another important target for early and long-term rapamycin effects.

Evaluation of disintegration in prevesical ureteral calculi by 3-dimensional endo-ultrasound with surface rendering.

PURPOSE: Lower ureteral calculi are accessible by transrectal endo-ultrasound, including 3-dimensional image processing with surface rendering. We analyzed the efficiency of this innovative technology compared with that of standard radiographic examinations with respect to the outcome of extracorporeal shock wave lithotripsy (ESWL) (Dornier Medical Systems, Marietta, Georgia). MATERIALS AND METHODS: In patients with prevesical or intramural calculi we performed excretory urography and transrectal endo-ultrasound using a Combison 530 device (Kretz Technik, Zipf, Austria) with integrated optional 3-dimensional image processing and surface rendering. A total of 102 ESWL cases (108 radiopaque stones, 145 ESWL sessions) were included in the study. One day after ESWL an evaluation was independently performed by a radiologist (plain x-ray) and a urologist (transrectal endo-ultrasound). Results were compared to the outcome determined during further followup. RESULTS: All stones were evaluable by endo-ultrasound with surface rendering, whereas in 27 cases the stone was hidden by extraureteral processes on followup radiography. Immediately after endo-ultrasound 8 calculi were passed. In 7 of the remaining 110 cases (6.4%) radiographic examination did not confirm sonographic findings. In these cases evaluation by surface rendering proved to be correct on followup. CONCLUSIONS: Endo-ultrasound with surface rendering proved to be highly effective for evaluating ESWL success in cases of prevesical ureteral stones. This technique is independent from bowel gas or other factors that impede radiological imaging. It is safe, easy to learn, well tolerated by patients and does not expose them to radiation.

Visualization of urinary stones by 3-D ultrasound with surface rendering.

The purpose of this study was to evaluate the diagnostic value of 3-D ultrasound with surface rendering in stones located in different parts of the urinary tract. A total of 55 patients with urinary stones were examined by transabdominal 3-D ultrasound (US) with surface rendering of the stone. In calculi of the prevesical ureter, bladder and prostatic urethra, transrectal 3-D endosonography was performed as well. The radiographic shape and the chemical analysis of the stone were correlated to the sonographic findings. The localization of the stone was: renal 23, ureteral 26, vesical 5 and urethral 1. All stones could be identified by 3-D US. Surface rendering produced useful results in stones > 2 cm with a 3.5-MHz transabdominal scanning probe, and in all stones that were accessible with a 7.5-MHz transrectal scanning probe. In stones < 1 cm, endoUS with surface rendering provides more information on the structure of the stone than plain radiography, but there is no correlation between surface and chemical analysis.

High-flow priapism: a combined interventional approach with angiography and colour Doppler.

Traumatic high-flow priapism caused by a pathologic influx from lacerated arteries to the cavernous bodies is usually treated by transcatheter arterial embolisation. The purpose of this study was to evaluate a combined interventional approach with intraoperative perineal colour Doppler ultrasound (US) while performing the embolisation procedure. Our aim was to reduce radiation exposure and application of iodinised contrast media. To achieve an optimal position of the angiography catheter directly before or in the fistula, instead of several x-ray examinations with contrast media, a perineal colour Doppler US examination was performed while saline or US contrast media were injected through the catheter. The flow pattern allows the assessment of the occlusion of the fistula and the intact flow in the arteries that were not damaged. In four patients (unilateral fistula: 1, bilateral fistulas: 3) with traumatic high-flow priapism, this technique was performed combined with a conventional angiographic control of the embolisation. Seven embolisation sessions were performed. Only in one case, a dislocation of a microcoil required a second session. In all cases, the priapism disappeared immediately after the final session, leading to a restored erectile function. This combined approach may reduce the exposure to radiation and contrast media, especially in children, who often suffer from multiple fistulas and need more than one session. The procedure also prevents an occlusion of intact arteries that affects the penile perfusion and increases the risk of erectile dysfunction.