HER2 copy number determination in breast cancer using the highly sensitive droplet digital PCR method.

Human epidermal growth factor receptor 2 (HER)-positive breast cancer (BC) is characterized by an aggressive clinical course. In the case of HER2 overexpression/amplification, patients benefit from HER2-targeting therapies. Standardized diagnostic HER2 assessment includes immunohistochemistry (IHC) and/or in situ hybridization (ISH). The aim of this study was to compare this "gold standard" with the Droplet Digital™ polymerase chain reaction (ddPCR), a method that allows sensitive and precise detection of copy number variations (CNV) in FFPE (formalin-fixed, paraffin-embedded) DNA samples. Partitioning of the PCR reaction into 20,000 droplets enables a precise quantitative "CN" discrimination also in heterogeneous samples. FFPE breast cancer samples (n = 170) with routinely assessed HER2 status by IHC/ISH were retrospectively analyzed using the ddPCR CNV ERBB2 assay. Comparison of HER2 status assessment by the two methods revealed concordant results in 92.9% (158/170) of the cases. Discrepant cases were verified and interpreted. For ddPCR, a cut off value of 3 HER2 copies was set to distinguish between HER2-negative and HER2-positive BC. Results obtained with the ddPCR CNV ERBB2 assay were consistent and reproducible, and serial dilutions demonstrated a high stability and sensitivity of the method. The ddPCR CNV ERBB2 assay may be a specific and convenient tool to quantify HER2 copy numbers in BC samples. In our study, this method showed high reproducibility in accuracy of HER2 assessment compared to IHC/ISH analysis.

Fetal Doppler monitoring during maternal open-heart surgery: Case report and key aspects of a multidisciplinary challenge.

CASE REPORT: Herein we present the case of a 33-year nulliparous woman at 21 weeks of gestation with mitral valve vegetation resulting from infective endocarditis. Due to the mother's critical condition caused by consecutive thromboembolic events, surgery with cardiopulmonary bypass was indicated. During surgery the fetus was monitored by a specialized obstetrician who repetitively measured the Doppler indices of the umbilical artery, Ductus venosus and uterine artery. Right after CO2 was insufflated into the operating area, the Doppler monitoring showed an increased Pulsatility Index of the Umbilical artery right before fetal distress with bradycardia occurred. A subsequent maternal arterial blood gas analysis showed an acidosis with hypercapnia. Consequently, the CO2 insufflation was stopped and the gas flow on the Heart Lung Machine increased. After regaining homeostasis of acidosis, the Doppler Indices and fetal heart rate recovered. The remaining surgery and postoperative course were uneventful. At the 37 weeks of gestation a healthy boy was delivered by Cesarean section and at the age of two years, the neurodevelopment was assessed, which indicated normal development in mental cognition, language and motoric. This report presents a periodic Doppler examination of the maternal and fetal circulation during surgery on CPB while also discussing the possible impact of fetal monitoring in managing open cardiac surgery in pregnancy.

Diagnosis and Therapy of Iron Deficiency Anemia During Pregnancy: Recommendation of the Austrian Society for Gynecology and Obstetrics (OEGGG).

This overview analyzes the data on the controversial therapy of iron substitution during pregnancy, the diagnosis of iron deficiency anemia and the indication-related therapy, and is the first recommendation issued by the OEGGG on the appropriate therapy. The effects of anemia during pregnancy on postnatal outcomes have been intensively investigated with heterogeneous results. A final scientific conclusion with regards to the "optimal" maternal hemoglobin level is limited by the heterogeneous results of various studies, many of which were conducted in emerging nations (with different dietary habits and structural differences in the respective healthcare systems). The current literature even suggests that there may be a connection between both decreased and increased maternal serum hemoglobin concentrations and unfavorable short-term and long-term neonatal outcomes. In Austria, 67 percent of pregnant women take pharmacological supplements or use a variety of dietary supplements. Clinically, the prevalence of maternal anemia is often overestimated, leading to overtreatment of pregnant women (iron substitution without a medical indication). To obtain a differential diagnosis, a workup of the indications for treatment should be carried out prior to initiating any form of iron substitution during pregnancy. If treatment is medically indicated, oral iron substitution is usually sufficient. Because of the restricted approval and potential side effects, medical indications for intravenous iron substitution should be limited. Intravenous iron substitution without a prior detailed diagnostic workup is an off-label use and should only be used in very limited cases, and women should be advised accordingly.

Tuning the Elasticity of Nanogels Improves Their Circulation Time by Evading Immune Cells.

Peptide receptor radionuclide therapy is used to treat solid tumors by locally delivering radiation. However, due to nephro- and hepato-toxicity, it is limited by its dosage. To amplify radiation damage to tumor cells, radiolabeled nanogels can be used. We show that by tuning the mechanical properties of nanogels significant enhancement in circulation half-life of the gel could be achieved. We demonstrate why and how small changes in the mechanical properties of the nanogels influence its cellular fate. Nanogels with a storage modulus of 37 kPa were minimally phagocytosed by monocytes and macrophages compared to nanogels with 93 kPa modulus. Using PET/CT a significant difference in the blood circulation time of the nanogels was shown. Computer simulations affirmed the results and predicted the mechanism of cellular uptake of the nanogels. Altogether, this work emphasizes the important role of elasticity even for particles that are inherently soft such as nano- or microgels.

Human Co- and Triple-Culture Model of the Alveolar-Capillary Barrier on a Basement Membrane Mimic.

The development of an in vitro model resembling the alveolar-capillary barrier might be a highly beneficial tool to study lung physiology as well as the immune response of the lung to infection or after exposure to nanoparticles. This study is based on an in vitro alveolar barrier developed on a basement membrane mimic, composed of ultrathin nanofiber meshes generated via electrospinning using bioresorbable poly(ɛ-caprolactone). As cellular components, NCI H441, resembling the alveolar epithelial cells, and ISO-HAS-1, an endothelial cell line, were used to perform bipolar coculture experiments for a total cultivation period of 14 days. In addition to immunohistochemical and immunofluorescent studies, transepithelial electrical resistance (TER) and transport capabilities of the in vitro model system were investigated. Alveolar barrier function could be clearly determined for the postulated bipolar coculture system on the basement membrane mimic, since TER increased during the course of bipolar cultivation. Furthermore, to gain first insights into possible lung inflammatory reactions in vitro, this coculture model was further expanded by a human leukemia monocyte cell line (THP-1). This triple-culture system was able to maintain adequately the barrier properties of the bipolar coculture, thus making this in vitro model consisting of epithelial, endothelial, and immune cells on a basement membrane mimic a promising basis for further studies in tissue engineering.

Intraoperative Electron Radiotherapy (IOERT) in the Treatment of Primary Breast Cancer.

IOERT (intraoperative electron radiotherapy) in breast cancer is used either as a boost (10-12 Gy) followed by whole breast irradiation (WBI) or as full-dose partial breast irradiation (PBI, 20-24 Gy) during breast-conserving surgery. IOERT has the longest evidence of all IORT techniques. When administered as a boost, excellent low local recurrence rates were observed in long-term follow-up >5 years. Even in high-risk groups like triple-negative or locally advanced breast cancers, IOERT contributes to long-term local control rates of more than 90%. For selected low-risk groups, IOERT as PBI with 21 Gy seems to be a viable treatment alternative to standard WBI. IOERT has been shown to be advantageous for several reasons: Geographic misses are avoided due to direct visualization of the tumor bed; thus, a high single dose is delivered with utmost precision to small volumes, completely sparing the skin and ensuring good long-term cosmetic outcome. Furthermore, high single doses seem to induce biological mechanisms with verifiable antitumor capability in in-vitro cell-line studies. In addition, IOERT markedly shortens the overall treatment time both in combination with (now mostly hypofractionated) WBI or as a PBI in selected low-risk constellations.

Intraoperative Tumor Bed Boost With Electrons in Breast Cancer of Clinical Stages I Through III: Updated 10-Year Results.

PURPOSE: To assess retrospectively the role of an anticipated intraoperative tumor electron radiation therapy (IOERT) as a bed boost during breast-conserving surgery followed by conventional whole breast irradiation (WBI). METHODS AND MATERIALS: An unselected cohort of 770 breast cancer patients of all risk types was analyzed in terms of local control (LC) and survival outcome. Patients were treated by breast-conserving surgery, IOERT of 10 Gy, and WBI to total median doses of 54 Gy (range, 1.6-2). Patients were retrospectively analyzed for LC, locoregional control, metastasis-free survival (MFS), overall survival (OS), and breast cancer-specific survival (BCSS). RESULTS: After a median follow-up of 121 months (range, 4-200), 21 (2.7%) in-breast recurrences (IBRs) were observed, 107 patients (14%) died and 106 (14%) developed metastases. Ten-year rates of LC, locoregional control, MFS, OS, and BCSS amounted to 97.2%, 96.5%, 86%, 85.7%, and 93.2 %, respectively. In multivariate analysis, HER2+ and triple-negative breast cancer subtype (TN) turned out to be significant negative predictors for IBRs (hazard ratios, 15.02 and 12.87, respectively; P < .05). Sorted by subtypes, 10-year LC rates were observed in 98.7% (range, 96.7%-99.5%) (luminal A), 98% (range, 94%-99.3%) (luminal B), 87.9% (range, 66.2%-96%) (HER2+), and 89% (range, 76.9%-94.9%) (TN), respectively. CONCLUSIONS: After 10 years, boost IOERT maintains high LC rates in any risk setting.

Mesonephric adenocarcinoma of the vagina : Diagnosis and multimodal treatment of a rare tumor and analysis of worldwide experience.

BACKGROUND: Mesonephric adenocarcinoma of the vagina is an extremely rare tumor of the female genital tract, with only a few cases reported so far worldwide. Consequently, there is no established standard treatment and limited knowledge about the prognosis and biologic behavior of vaginal mesonephric adenocarcinoma. METHODS: This report documents a new case of vaginal mesonephric adenocarcinoma diagnosed in a 54-year-old woman, and analyzes this in the context of all previously published cases. RESULTS: MRI demonstrated that the 2.5 × 1.8 cm tumor of the vaginal wall was invading urethra and bladder. Following surgical excision, histologic analysis determined mesonephric adenocarcinoma of the vagina, stage pT2 R1. In order to avoid the mutilating extended surgery which would be required to reach R0 and considerable impairment of quality of life, adjuvant radiochemotherapy was administered with external radiation and brachytherapy, including 5 cycles of cisplatin (40 mg/m²) for radiosensitization. After 4 years of continuous oncologic follow-up, the patient is alive and clinically free of disease. CONCLUSION: In this case it was shown that adjuvant radiochemotherapy with radiation and brachytherapy was effective to manage the surgical R1 situation and maintain the patient's life quality. More published cases reports are needed to gradually substantiate optimal treatment strategies.

Hsa-miR-375 is a predictor of local control in early stage breast cancer.

BACKGROUND: A long-term analysis by the Early Breast Cancer Trialist Group (EBCTG) revealed a strong correlation between local control and cancer-specific mortality. MicroRNAs (miRs), short (20-25 nucleotides) non-coding RNAs, have been described as prognosticators and predictors for breast cancer in recent years. The aim of the current study was to identify miRs that can predict local control after breast conserving therapy (BCT) in early stage breast cancer. RESULTS: Clinical data of 46 early stage breast cancer patients with local relapse after BCT were selected from the institutional database. These patients were matched to 101 control patients showing identical clinical features but without local relapse. The study was conducted in two steps. (1) In the pilot study, 32 patients (16 relapses versus 16 controls) were screened for the most de-regulated microRNAs (= candidate microRNAs) in a panel of 1250 miRs by microarray technology. Eight miRs were found to be significantly de-regulated. (2) In the validation study, the candidate microRNAs were analyzed in an independent cohort of 115 patients (30 relapses versus 85 controls) with reverse transcription quantitative polymerase chain reaction (RT-qPCR). From these eight candidates, hsa-miR-375 could be validated. Its median fold change was 2.28 (Mann-Whitney U test, corrected p value = 0.008). In the log-rank analysis, high expression levels of hsa-miR-375 correlated with a significantly higher risk of local relapse (p = 0.003). In a multivariate analysis (forward stepwise regression) including established predictors and prognosticators, hsa-miR-375 was the only variable that was able to distinguish the statistical significance between relapse and control groups (raw p value = 0.000195 HR = 0.76, 95 % CI 0.66-0.88; corrected p value = 0.005). CONCLUSIONS: Hsa-miR-375 predicts local control in patient with early stage breast cancer, especially in estrogen receptor α (ER-α)-positive patients. It can therefore serve as an additional molecular marker for treatment choice independently from known predictors and prognosticators. Validation in larger prospective studies is warranted.

Survival and local control rates of triple-negative breast cancer patients treated with boost-IOERT during breast-conserving surgery.

AIM: The purpose of this work was to retrospectively evaluate survival and local control rates of triple-negative breast cancer subtypes classified as five marker negative (5NP) and core basal (CB), respectively, after breast-conserving surgery and intraoperative boost radiotherapy with electrons (IOERT) followed by whole breast irradiation. METHODS AND MATERIALS: A total of 71 patients with triple-negative breast cancer were enrolled, who were treated with lumpectomy, axillary lymph node dissection, and IOERT with 9.6 Gy (median Dmax) followed by normofractionated whole breast irradiation to median total doses of 54 Gy. Chemotherapy was applied in a neoadjuvant (12 %), adjuvant (75 %), or combinational setting (7 %). RESULTS: After a median follow-up of 97 months (range 4-170 months), 5 in-breast recurrences were detected (7.0 %). For all patients, 8-year actuarial rates for local control, metastases-free survival, disease-specific survival, and overall survival amounted to 89, 75, 80, and 69 %, respectively. All local recurrences occurred in grade 3 (G3) tumors irrespective of their specific immunohistochemical phenotype; thus, the local control rate for grades 1/2 (G1/2) was 100 % for both 5NP and CB, while for G3 it was 88 % for 5NP and 90 % for CB (p = 0.65 and 0.82, respectively, n.s.). For disease-specific survival, only the difference of the best-prognosis group 5-NP/G3 vs. the worst-prognosis cohort CB/G1/2 was statistically significant: 90 % vs. 54 % (p = 0.03). CONCLUSION: Boost-IOERT provides acceptable long-term in-breast control in triple negative breast cancer. The best subgroup in terms of disease-specific survival was represented by 5NP in combination with tumor grading G3.

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

BACKGROUND: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. METHODS: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. RESULTS: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. CONCLUSIONS: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

Cutaneous manifestations in trisomy 13 mosaicism: A rare case and review of the literature.

Trisomy 13 mosaicism is a rare genetic disorder affecting a small minority of all trisomy 13 cases. It occurs when two cell populations that are karyotypically different are present in the same individual and are derived from a single zygote. As a rule, the phenotype is mitigated to a less dysmorphic appearance and longer survival, making genetic counseling a difficult task. Capillary hemangiomas are a common feature of full trisomy 13, seen in 27-56% of all cases. We report on an 18-months-old girl with extensive cutaneous anomalies, mild dysmorphic features, and slight psychomotor delay, without structural defects and provide an up-to-date review of all cases of trisomy 13 mosaicism with skin involvement. To our knowledge, this is the second clinical report of a patient with trisomy 13 mosaicism with hemangiomas and port wine stains, but no structural defects. © 2015 Wiley Periodicals, Inc.

IOERT as anticipated tumor bed boost during breast-conserving surgery after neoadjuvant chemotherapy in locally advanced breast cancer--results of a case series after 5-year follow-up.

To evaluate retrospectively rates of local (LCR) and locoregional tumor control (LRCR) in patients with locally advanced breast cancer (LABC) who were treated with preoperative chemotherapy (primary systemic treatment, PST) followed by breast-conserving surgery (BCS) and either intraoperative radiotherapy with electrons (IOERT) preceding whole-breast irradiation (WBI) (Group 1) or with WBI followed by an external tumor bed boost (electrons or photons) instead of IOERT (Group 2). From 2002 to 2007, 83 patients with clinical Stage II or III breast cancer were enrolled in Group 1 and 26 in Group 2. All patients received PST followed by BCS and axillary lymph node dissection. IOERT boosts were applied by single doses of 9 Gy (90% reference isodose) versus external boosts of 12 Gy (median dose range, 6-16) in 2 Gy/fraction (ICRU). WBI in both groups was performed up to total doses of 51-57 Gy (1.7-1.8 Gy/fraction). The respective median follow-up times for Groups 1 and 2 amount 59 months (range, 3-115) and 67.5 months (range, 13-120). Corresponding 6-year rates for LCR, LRCR, metastasis-free survival, disease-specific survival and overall survival were 98.5, 97.2, 84.7, 89.2 and 86.4% for Group 1 and 88.1, 88.1, 74, 92 and 92% for Group 2, respectively, without any statistical significances. IOERT as boost modality during BCS in LABC after PST shows a trend to be superior in terms of LCR and LRCR in comparison with conventional boosts.

Local foreign body reaction of peritoneum after rupture of cystic partially immature teratoma.

This is a pictorial report of rare sequelae after an unintended intraoperative rupture of a cystic teratoma. A 30-year-old woman was operated on for a mature cystic teratoma of the right ovary with an unintended intraoperative rupture of the ovarian tumor during the procedure. Postoperatively, the final immune histologic report showed partial neuroendocrine differentiation of an immature origin. At relaparoscopy for staging 7 weeks later, several suspicious peritoneal lesions of up to a 2.5-cm diameter were discovered and excised for which malignancy could not be excluded macroscopically. However, the final histologic report revealed a foreign body reaction related to spilling of the content of the mature teratoma. It is important to distinguish local peritoneal reaction from chemical peritonitis. The postoperative follow-up regarding symptomatic recurrence was uneventful.

Health economic impact of risk group selection according to ASCO-recommended biomarkers uPA/PAI-1 in node-negative primary breast cancer.

Invasion factors uPA/PAI-1 are guideline-recommended (ASCO, AGO) biomarkers for decision support regarding adjuvant chemotherapy (CTX) in women with primary breast cancer. They define a high-risk group with strong benefit from adjuvant CTX and a low-risk group with uncertain benefit and excellent survival without CTX. In a target population (age > 35/N0/G2/HR+/HER2-), administration of adjuvant CTX is not mandatory in Germany and other countries. Based on existing data, this economic model was developed to determine for the first time health economic impact of uPA/PAI-1 testing. Incremental cost-effectiveness ratio (ICER) resulting from uPA/PAI-1 testing was estimated for the target population by Markov modeling and sensitivity analysis. Survival data, CTX-uPA/PAI-1 interactions, and uPA/PAI-1 hazard ratios were derived from the Chemo N0 trial and other evidence. Incremental costs were computed from a payer's perspective appropriate to the German setting. Incremental effectiveness in life years (ly) was estimated taking into account age-adjusted life expectancy, disease-free survival (with/without CTX), and 2 years post-relapse survival. Sensitivity analysis was performed by varying residual adjuvant CTX benefit in the low-risk group, denoted HR_CTX(LR), in range 0.8-0.99. All patients receive adjuvant endocrine therapy. Test is restricted to patients willing to forgo CTX if both markers are below specific cut-off values and to undergo CTX otherwise. For a typical 55-year-old patient, comparing to an "all-CTX" strategy without the test, ICER (all-CTX vs. test) > 50,000 if HR_CTX(LR) > 0.85, with savings of 18,500 per low-risk patient attributable to the test. The cost-effectiveness of forgoing CTX is very high as HR_CTX(LR) approaches one. Conversely, comparing to a "no-CTX" strategy (e.g., patients who initially refuse CTX) without the test, the test is very cost-effective at all ages in the target group if high-risk patients are willing to undergo CTX: ICER (test vs. no-CTX) < 6,000 at age 55 and even better at younger ages, remaining < 25,000 up to age 75. The main determinants of cost utility are age and residual CTX benefit in low-uPA/PAI-1 patients. The uPA/PAI-1 test is cost-effective in the target group compared to either an "all-CTX" or a "no-CTX" scenario. This model thus lends health economic support to current guideline recommendations that uPA/PAI-1 testing is beneficial for BC patients with no lymph node involvement.

Relevance of health economics in breast cancer treatment: integration of economics in the management of breast cancer at the clinic level.

Since the introduction of the diagnosis-related groups (DRG) system with cost-related and entity-specific flat-rate reimbursements for all in-patients in 2004 in Germany, economics have become an important focus in medical care, including breast centers. Since then, physicians and hospitals have had to gradually take on more and more financial responsibilities for their medical care to avoid losses for their institutions. Due to financial limitations of resources, most medical services have to be adjusted to correlating revenues, which results in the development of a variety of active measures to understand, steer, and optimize costs, resources and related processes for breast cancer treatment. In this review, the challenging task to implement microeconomic management at the clinic level for breast cancer treatment is analyzed from breast cancer-specific publications. The newly developed economic management perspective is identified for different stakeholders in the healthcare system, and successful microeconomic projects and future aspects are described.

Stillbirth in week 19 of pregnancy followed by maternal death as a consequence of refused chemotherapy for non-hodgkin's lymphoma--significance of adjuvant chemotherapy in women of reproductive age.

BACKGROUND: Due to rising cure rates in cancer, the question of preserving fertility in young female patients becomes more important. Especially in lymphomas, incidence and long-time survival have increased. Hematologists and gynecologists have to treat more and more female patients who wish to become pregnant despite their disease and/or after finishing treatment. CASE REPORT: We report on a 28-year-old patient with highly malignant non-Hodgkin's lymphoma (peripheral T cell lymphoma, Ann Arbor stage IV) and main manifestation at the gastric antrum, with a distinct wish for becoming pregnant. Chemotherapy was strongly recommended to her, but she refused. After she had conceived, the disease recurred, followed by stillbirth in week 19 of gestation and death due to gastric perforation and septic shock. CONCLUSIONS: Facing the risk of sterility after chemotherapy should not induce patients to refuse chemotherapy and risk their lives. Treatment of young female cancer patients should therefore always include a thorough discussion about other ways of preserving fertility for the time after treatment. Such strategies exist, although their success is still limited and not every patient is eligible for them.

Advanced metastatic melanoma during pregnancy: a multidisciplinary challenge.

BACKGROUND: Metastatic melanoma during pregnancy represents a life-threatening situation not only for the mother but also for the fetus due to aggressive therapy and potential maternal-fetal metastasis. CASE REPORT: We report the case of a 37-year-old woman with advanced metastatic malignant melanoma during her first pregnancy, with a review of the literature. In this case, a tight and primarily interdisciplinary obstetrical and dermatological case management enabled the delivery of a small but healthy premature infant in the 29th week of gestation by planned Cesarean section. However, due to progressive disease, the mother died only 10 weeks after the delivery of the baby. CONCLUSION: Sufficient perinatal and oncologic experience provided, diagnostic and surgical interventions as well as radiotherapy and chemotherapy in metastatic melanoma disease are feasible and relatively safe even during pregnancy.

Purple membranes as microscaled nanopatterned biosubstrates for reversible attachment of biocomponents.

One of the key challenges in the construction of complex bionanotechnological building blocks and devices is the controlled linkage and release of biomolecular components to and from a biomolecular surface. Here we report a versatile, easy and universally applicable method for the reversible assembly of protein-protein conjugates. The process is demonstrated using green fluorescent protein (GFP) and purple membranes (PM) as model compounds. GFP was reversibly bound to PM patches which served as a biomolecular model surface. Due to its size in the micrometre range PM is, as far as its Brownian diffusion is considered, like a solid surface. PMs comprising the mutated bacteriorhodopsin BR-D36C were employed, where aspartic acid in position 36 was replaced with cysteine. The introduced cysteine is accessible from the cytoplasmic side of the membrane. The cysteine group was first functionalized with a nitrilotriacetic acid group (NTA) and then, after loading with Ni, histidine-tagged GFP was bound to the chemically modified PM surface the well-known NTA-His complex. Binding and release of GFP from the PM surface was monitored by atomic force microscopy (AFM).