Glucagon-like peptide-1 and glucagon-like peptide-2 regulation during human liver regeneration.

Accumulating evidence suggests that metabolic demands of the regenerating liver are met via lipid metabolism and critical regulators of this process. As such, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) critically affect hepatic regeneration in rodent models. The present study aimed to evaluate potential alterations and dynamics of circulating GLP-1 and GLP-2 in patients undergoing liver resections, focusing on post-hepatectomy liver failure (PHLF). GLP-1, GLP-2, Interleukin-6 (IL-6) and parameters of lipid metabolism were determined perioperatively in fasting plasma of 46 patients, who underwent liver resection. GLP-1 and GLP-2 demonstrated a rapid and consistently inverse time course during hepatic regeneration with a significant decrease of GLP-1 and increase of GLP-2 on POD1. Importantly, these postoperative dynamics were significantly more pronounced when PHLF occurred. Of note, the extent of resection or development of complications were not associated with these alterations. IL-6 mirrored the time course of GLP-2. Assessing the main degradation protein dipeptidyl peptidase 4 (DPP4) no significant association with either GLP-1 or -2 could be found. Additionally, in PHLF distinct postoperative declines in plasma lipid parameters were present and correlated with GLP-2 dynamics. Our data suggest dynamic inverse regulation of GLP-1 and GLP-2 during liver regeneration, rather caused by an increase in expression/release than by changes in degradation capacity and might be associated with inflammatory responses. Their close association with circulating markers of lipid metabolism and insufficient hepatic regeneration after liver surgery suggest a critical involvement during these processes in humans.

Liver surgery in the 2020s: ante-situm and in-situ resection are still indicated - A single-center study.

BACKGROUND: Tumor infiltration of the hepatic outflow comprising all three hepatic veins and the inferior vena cava remains a surgical challenge. Liver resection under total vascular exclusion with or without extracorporeal bypass has been described as a therapeutic option for these tumors. Here, we present our experience with these complex surgical methods. METHODS: We searched our database for patients treated with an in-situ or ante-situm liver resection (ISR and ASR, respectively) with extracorporeal bypass. We collected demographic and perioperative data. RESULTS: From January 2010 to December 2021, we performed 2122 liver resections. Nine patients were treated with ASR and five were treated with ISR. Out of these 14 patients, six had colorectal liver metastases, six had cholangiocarcinoma, and two had non-colorectal liver metastases. The median operative time and bypass time in all patients were 536.5 and 150 min, respectively. Compared with ISR, ASR required a longer operative time (ASR 586 min and ISR 495 min) and a longer bypass time (ASR 155 min and ISR 122 min). Morbidity (Clavien-Dindo grade > 3A adverse events) occurred in 78.5% of patients. 90-day postoperative mortality was 7%. Median overall survival was 33 months. Seven patients experienced recurrence. In these patients, median disease-free survival was 9 months. CONCLUSION: Resection of tumors infiltrating the hepatic outflow poses a high risk for patients. However, with rigorous selection and an experienced perioperative team, these patients can be treated surgically with reasonable oncological outcomes.

Neoadjuvant immunotherapy in gastrointestinal cancers - The new standard of care?

The development of immune checkpoint inhibitors (ICI) offers novel treatment possibilities for solid cancers, with the crucial benefit of providing higher cure rates. These agents have become part of standard treatments in the metastatic and adjuvant setting for select cancers, such as melanoma, non-small cell lung cancer (NSCLC) or urological malignancies. Currently, there is ample clinical interest in employing ICI in a neoadjuvant setting with a curative intent. This approach is especially supported by the scientific rationale that ICI primarily stimulate the host immune system to eradicate tumor cells, rather than being inherently cytotoxic. Aside from tumor downstaging, neoadjuvant immunotherapy offers the potential of an in situ cancer vaccination, leading to a systemic adjuvant immunological effect after tumor resection. Moreover, preclinical data clearly demonstrate a synergistic effect of ICI with radiotherapy (RT), chemoradiotherapy (CRT) or chemotherapy (ChT). This review harmonizes preclinical concepts with real world data (RWD) in the field of neoadjuvant ICI in gastrointestinal (GI) cancers and discusses their limitations. We believe this is a crucial approach, since up to now, neoadjuvant strategies have been primarily developed by clinicians, whereas the advances in immunotherapy primarily originate from preclinical research. Currently there is limited published data on neoadjuvant ICI in GI cancers, even though neoadjuvant treatments including RT, CRT or ChT are frequently employed in locally advanced/oligometastatic GI cancers (i.e. rectal, pancreatic, esophagus, stomach, etc.). Utilizing established therapies in combination with ICI provides an abundance of opportunities for innovative treatment regimens to further improve survival rates.

Immune checkpoints and liver resection after neoadjuvant chemotherapy including bevacizumab in patients with microsatellite-stable colorectal liver metastases.

BACKGROUND: The clinical value of immune checkpoint expression as prognostic biomarker in bevacizumab-pretreated patients with resected microsatellite-stable (MMS) colorectal liver metastases is unclear and was retrospectively investigated in this study. METHODS: Expression analyses of IDO-1, PD-L1, and CTLA-4 were performed by immunohistochemistry in resected bevacizumab-pretreated colorectal liver metastases. Association of immune checkpoint expression in tumor cells and immune cells with response and clinical outcome was investigated. Expression profiles were compared with those of patients with anti-EGFR-targeted therapy and lung metastases, respectively. RESULTS: One hundred thirty-six patients with MMS disease were investigated (79 (58.1%) male/57 (41.9%) female, median age 62.9 years (range 31.0-80.4)). High expression of IDO-1 in immune cells was associated with longer OS (not reached versus 44.8 months, HR 0.23 (95% CI 0.09, 0.55), P = 0.001). Low expression of CTLA-4 in tumor cells was associated with better histological response (26 major, 19 partial, 18 none versus 14 major, 23 partial, 30 none, P = 0.032). Expression profiles differed compared to patients with anti-EGFR-targeted therapy and patients with lung metastases. CONCLUSION: Immune checkpoint expression was associated with response and survival. IDO-1 may serve as a novel prognostic and/or predictive biomarker in patients with MMS colorectal liver metastases.

Prognosis and Circumferential Margin in Patients with Resected Hilar Cholangiocarcinoma.

BACKGROUND: Resection margin status is a known prognosticator in patients who undergo resection for hilar cholangiocarcinoma. However, the influence of an isolated positive circumferential margin on clinical outcome is unclear. METHODS: Patients with resected de novo hilar cholangiocarcinoma from two European hepatobiliary centres (Medical University of Vienna and Aintree University Hospital, 2006-2016) were classified according to resection margin status (negative, surgically positive, isolated circumferentially positive) and investigated with respect to overall survival (OS), recurrence-free survival (RFS) and recurrence pattern. RESULTS: Eighty-three (48 male/35 female) patients were enrolled. The median age was 64 years (range 33-80). The median follow-up was 21.7 months (range 0.3-92.4). Forty (48%) patients had negative resection margins, 25 (30%) had an isolated positive circumferential margin and 18 (22%) had a positive surgical margin. The 5-year OS rates in patients with negative, isolated positive circumferential and positive surgical resection margins were 47%, 33% and 0%, respectively. Median OS was 45.6, 32.7 and 14.5 months, respectively (log rank, P = 0.011). Upon multivariable Cox regression analysis, resection margin status and lymph node status remained statistically significant (P < 0.05). No difference with respect to RFS and recurrence pattern was found between the groups (P > 0.05). CONCLUSION: Our data show that these three resection margin types were associated with different clinical outcomes. Circumferential margin status may therefore serve as a novel prognostic biomarker.

Short-course radiotherapy promotes pro-inflammatory macrophages via extracellular vesicles in human rectal cancer.

BACKGROUND: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. METHODS: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. RESULTS: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. CONCLUSIONS: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies.

From threat to cure: understanding of virus-induced cell death leads to highly immunogenic oncolytic influenza viruses.

Oncolytic viruses constitute an emerging strategy in immunomodulatory cancer treatment. The first oncolytic virus, Talimogene laherparepvec (T-VEC), based on herpes simplex virus 1 (HSV-1), was approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) in 2015. The field of oncolytic virotherapy is still in its beginnings, since many promising viruses remain only superficially explored. Influenza A virus causes a highly immunogenic acute infection but never leads to a chronic disease. While oncolytic influenza A viruses are in preclinical development, they have not made the transition into clinical practice yet. Recent insights into different types of cell death caused by influenza A virus infection illuminate novel possibilities of enhancing its therapeutic effect. Genetic engineering and experience in influenza A virus vaccine development allow safe application of the virus in patients. In this review we give a summary of efforts undertaken to develop oncolytic influenza A viruses. We discuss strategies for targeting viral replication to cancerous lesions and arming them with immunogenic transgenes. We furthermore describe which modes of cell death are induced by influenza A virus infection and how these insights may be utilized to optimize influenza A virus-based oncolytic virus design.

Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis.

BACKGROUND: The monoclonal antibody (mAb) trastuzumab is part of the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer. Antibody-dependent cell-mediated phagocytosis (ADCP) and cytotoxicity (ADCC) are major mechanisms of action of the mAb trastuzumab. Histone deacetylase inhibitors (HDACi), such as valproic acid (VPA) or vorinostat (SAHA), exert several immunostimulatory properties, which contribute at least in part to their anticancer effect. However, the impact of HDACi-induced immunostimulatory effects on trastuzumab-mediated anti-tumor immune response is not well characterized. METHODS: We analyzed the ADCP and ADCC activity of peripheral blood mononuclear cells (PBMCs) from age and gender-matched healthy volunteers (n=5) against HDACi-treated HER2-overexpressing breast cancer cells (SKBR3), using a well-established in vitro three-color imaging flow cytometry and flow cytometry approach. RESULTS: VPA and SAHA enhanced trastuzumab-mediated ADCP and trastuzumab-independent cytotoxicity. Mechanistically, VPA upregulated the activating antibody-binding receptor Fc-gamma receptor (FcγR) IIA (CD32A) on monocytes (CD14+). Moreover, VPA and SAHA downregulated the anti-apoptotic protein myeloid leukemia cell differentiation 1 (MCL1) in breast cancer cells. Additionally, VPA and SAHA induced an immunogenic cell death, characterized by the exposure of calreticulin (CALR), as well as decreased the "do not eat me" signal CD47 on tumor cells. CONCLUSIONS: HDACi VPA and SAHA increase trastuzumab-mediated phagocytosis and trastuzumab-independent cytotoxicity. The immunomodulatory activities of those HDACi support a rationale combined treatment approach with mAb for cancer treatment.

Surgical Complexity and Outcome During the Implementation Phase of a Robotic Colorectal Surgery Program-A Retrospective Cohort Study.

BACKGROUND: Robotic surgery holds particular promise for complex oncologic colorectal resections, as it can overcome many limitations of the laparoscopic approach. However, similar to the situation in laparoscopic surgery, appropriate case selection (simple vs. complex) with respect to the actual robotic expertise of the team may be a critical determinant of outcome. The present study aimed to analyze the clinical outcome after robotic colorectal surgery over time based on the complexity of the surgical procedure. METHODS: All robotic colorectal resections (n = 85) performed at the Department of Surgery, Medical University of Vienna, between the beginning of the program in April 2015 until December 2019 were retrospectively analyzed. To compare surgical outcome over time, the cohort was divided into 2 time periods based on case sequence (period 1: patients 1-43, period 2: patients 44-85). Cases were assigned a complexity level (I-IV) according to the type of resection, severity of disease, sex and body mass index (BMI). Postoperative complications were classified using the Clavien-Dindo classification. RESULTS: In total, 47 rectal resections (55.3%), 22 partial colectomies (25.8%), 14 abdomino-perineal resections (16.5%) and 2 proctocolectomies (2.4%) were performed. Of these, 69.4% (n = 59) were oncologic cases. The overall rate of major complications (Clavien Dindo III-V) was 16.5%. Complex cases (complexity levels III and IV) were more often followed by major complications than cases with a low to medium complexity level (I and II; 25.0 vs. 5.4%, p = 0.016). Furthermore, the rate of major complications decreased over time from 25.6% (period 1) to 7.1% (period 2, p = 0.038). Of note, the drop in major complications was associated with a learning effect, which was particularly pronounced in complex cases as well as a reduction of case complexity from 67.5% to 45.2% in the second period (p = 0.039). CONCLUSIONS: The risk of major complications after robotic colorectal surgery increases significantly with escalating case complexity (levels III and IV), particularly during the initial phase of a new colorectal robotic surgery program. Before robotic proficiency has been achieved, it is therefore advisable to limit robotic colorectal resection to cases with complexity levels I and II in order to keep major complication rates at a minimum.

DNA damage predicts prognosis and treatment response in colorectal liver metastases superior to immunogenic cell death and T cells.

Preclinical models indicate that DNA damage induces type I interferon (IFN), which is crucial for the induction of an anti-tumor immune response. In human cancers, however, the association between DNA damage and an immunogenic cell death (ICD), including the release and sensing of danger signals, the subsequent ER stress response and a functional IFN system, is less clear. Methods: Neoadjuvant-treated colorectal liver metastases (CLM) patients, undergoing liver resection in with a curative intent, were retrospectively enrolled in this study (n=33). DNA damage (γH2AX), RNA and DNA sensors (RIG-I, DDX41, cGAS, STING), ER stress response (p-PKR, p-eIF2α, CALR), type I and type II IFN- induced proteins (MxA, GBP1), mature dendritic cells (CD208), and cytotoxic and memory T cells (CD3, CD8, CD45RO) were investigated by an immunohistochemistry whole-slide tissue scanning approach and further correlated with recurrence-free survival (RFS), overall survival (OS), radiographic and pathologic therapy response. Results: γH2AX is a negative prognostic marker for RFS (HR 1.32, 95% CI 1.04-1.69, p=0.023) and OS (HR 1.61, 95% CI 1.23-2.11, p<0.001). A model comprising of DDX41, STING and p-PKR predicts radiographic therapy response (AUC=0.785, p=0.002). γH2AX predicts prognosis superior to the prognostic value of CD8. CALR positively correlates with GBP1, CD8 and cGAS. A model consisting of γH2AX, p-eIF2α, DDX41, cGAS, CD208 and CD45RO predicts pathological therapy response (AUC=0.944, p<0.001). Conclusion: In contrast to preclinical models, DNA damage inversely correlated with ICD and its associated T cell infiltrate and potentially serves as a therapeutic target in CLM.

Mutational profile of colorectal cancer lung metastases and paired primary tumors by targeted next generation sequencing: implications on clinical outcome after surgery.

BACKGROUND: Pulmonary metastasectomy is one of the cornerstones in the treatment of oligometastatic colorectal cancer (CRC). However, the selection of patients who benefit from a surgical resection is difficult. Mutational profiling has become an essential part of diagnosis and treatment of malignant disease. Despite this, comprehensive data on the mutational profile of CRC and its clinical impact in the context of pulmonary metastasectomy is sparse. We therefore aimed to provide a complete mutational status of CRC pulmonary metastases (PM) and corresponding primary tumors by targeted next-generation sequencing (tNGS), and correlate sequencing data with clinical outcome variables. METHODS: Case-matched, formalin-fixed paraffin embedded surgical specimens of lung metastases (n=47) and matched primary CRC (n=24) were sequenced using the TruSeq Amplicon Cancer Panel (Illumina platform). Penalized Cox regression models were applied to identify mutations with prognostic impact. RESULTS: Mutations were found most frequently in APC, TP53 and KRAS, in both PM and matched primary tumors. Concordance between primary tumors and PM was 83.5%. Adaptive elastic-net regularized Cox regression models identified mutations being prognostic for time to pulmonary recurrence (EGFR, GNAQ, KIT, MET, and PTPN11) and for overall survival (OS) (PDGFRA, SMARCB1, and TP53). CONCLUSIONS: Our findings suggest that CRC PM harbor a variety of conserved and de novo mutations. We could identify a mutational profile predicting clinical outcome after pulmonary metastasectomy. Moreover, our data provide a rationale for future targeted therapies of patients with CRC lung metastases.

Exploring Metabolic Configurations of Single Cells within Complex Tissue Microenvironments.

Over the past years, plenty of evidence has emerged illustrating how metabolism supports many aspects of cellular function and how metabolic reprogramming can drive cell differentiation and fate. Here, we present a method to assess the metabolic configuration of single cells within their native tissue microenvironment via the visualization and quantification of multiple enzymatic activities measured at saturating substrate conditions combined with subsequent cell type identification. After careful validation of the approach and to demonstrate its potential, we assessed the intracellular metabolic configuration of different human immune cell populations in healthy and tumor colon tissue. Additionally, we analyzed the intercellular metabolic relationship between cancer cells and cancer-associated fibroblasts in a breast cancer tissue array. This study demonstrates that the determination of metabolic configurations in single cells could be a powerful complementary tool for every researcher interested to study metabolic networks in situ.

Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Viruses shape a diversity of ecosystems by modulating their microbial, eukaryotic, or plant host metabolism. The complexity of virus-host interaction networks is progressively fathomed by novel metagenomic approaches. By using a novel metagenomic method, we explored the virome in mammalian cell cultures and clinical samples to identify an extensive pool of mobile genetic elements in all of these ecosystems. Despite aseptic treatment, cell cultures harbored extensive and diverse phage populations with a high abundance of as yet unknown and uncharacterized viruses (viral dark matter). Unknown phages also predominated in the oropharynx and urine of healthy individuals and patients infected with cytomegalovirus despite demonstration of active cytomegalovirus replication. The novelty of viral sequences correlated primarily with the individual evaluated, whereas relative abundance of encoded protein functions was associated with the ecologic niches probed. Together, these observations demonstrate the extensive presence of viral dark matter in human and artificial ecosystems.-Thannesberger, J., Hellinger, H.-J., Klymiuk, I., Kastner, M.-T., Rieder, F. J. J., Schneider, M., Fister, S., Lion, T., Kosulin, K., Laengle, J., Bergmann, M., Rattei, T., Steininger, C. Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Human cytomegalovirus infection downregulates vitamin-D receptor in mammalian cells.

Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12h [hrs] post infection [p.i.] to 12% relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96h. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3% by 12h p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970% probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

Oncolytic influenza A virus expressing interleukin-15 decreases tumor growth in vivo.

BACKGROUND: Interleukin-15 has become a promising molecule in the context of eliciting an effective, antitumor immune response because it is able to stimulate cells of the innate and adaptive immune system. METHODS: We generated an interleukin-15-expressing oncolytic influenza A virus for the treatment of an established murine tumor model. RESULTS: Our oncolytic influenza A virus produced large amounts of interleukin-15 and induced proliferation and activation of human T cells in vitro. Intraperitoneal administration increased the amount of mouse natural killer cells and effector memory T cells, as well as T cell reactivity in vivo. Moreover, intratumoral injection induced a profound decrease in growth of established tumors in mice and increased the amount of tumor-infiltrating T cells and natural killer cells. CONCLUSION: We established a stable, IL-15-producing oncolytic influenza A virus with promising immunostimulatory and antitumor attributes.

Interleukin-24 inhibits influenza A virus replication in vitro through induction of toll-like receptor 3 dependent apoptosis.

New anti-viral agents and strategies are urgently needed to fight rapidly mutating viruses, as vaccine programs cannot react fast enough to prevent pandemics. Recently, we have shown that interleukin-24 (IL-24) sensitizes tumor cells to toll-like receptor 3 (TLR3) mediated apoptosis. As influenza A virus stimulates the TLR3 receptor, we hypothesized that IL-24 might also exert an anti-viral effect. This study demonstrates that IL-24 reduces the titer of different influenza A virus subtypes independently of type I interferon in an apoptosis dependent manner. The anti-viral effect of IL-24 correlated with caspase-3 activation and could be blocked by a pan-caspase inhibitor and by small interfering RNA (siRNA) directed towards TLR3. Surprisingly, caspase-3 activation in influenza A virus/IL-24-stimulated cells correlated with the down-regulation of the B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia 1 (Mcl-1). Correspondingly, knockdown of Mcl-1 by siRNA enhanced caspase activation in influenza A virus infected cells and was furthermore linked to a reduction of viral titers. We conclude that IL-24 exerts an anti-viral role selectively purging virally infected cells by leading to a down-regulation of Mcl-1. Our findings might therefore represent the first step towards a new rational concept in the development of anti-viral strategies based on the induction of apoptosis.

Trastuzumab mediates antibody-dependent cell-mediated cytotoxicity and phagocytosis to the same extent in both adjuvant and metastatic HER2/neu breast cancer patients.

BACKGROUND: Monoclonal antibodies (mAb), such as trastuzumab are a valuable addition to breast cancer therapy. Data obtained from neoadjuvant settings revealed that antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanism of action for the mAb trastuzumab. Conflicting results still call into question whether disease progression, prolonged treatment or concomitant chemotherapy influences ADCC and related immunological phenomena. METHODS: We analyzed the activity of ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) of peripheral blood mononuclear cells (PBMCs) from human epidermal growth factor receptor 2 (HER2/neu) positive breast cancer patients receiving trastuzumab therapy either in an adjuvant (n = 13) or metastatic (n = 15) setting as well as from trastuzumab treatment-naive (t-naive) HER2/neu negative patients (n = 15). PBMCs from healthy volunteers (n = 24) were used as controls. ADCC and ADCP activity was correlated with the expression of antibody binding Fc-gamma receptor (FcγR)I (CD64), FcγRII (CD32) and FcγRIII (CD16) on CD14+ (monocytes) and CD56+ (NK) cells, as well as the expression of CD107a+ (LAMP-1) on CD56+ cells and the total amount of CD4+CD25+FOXP3+ (Treg) cells. In metastatic patients, markers were correlated with progression-free survival (PFS). RESULTS: ADCC activity was significantly down regulated in metastatic, adjuvant and t-naive patient cohorts as compared to healthy controls. Reduced ADCC activity was inversely correlated with the expression of CD107a on CD56+ cells in adjuvant patients. ADCC and ADCP activity of the patient cohorts were similar, regardless of treatment duration or additional chemotherapy. PFS in metastatic patients inversely correlated with the number of peripheral Treg cells. CONCLUSION: The reduction of ADCC in patients as compared to healthy controls calls for adjuvant strategies, such as immune-enhancing agents, to improve the activity of trastuzumab. However, efficacy of trastuzumab-specific ADCC and ADCP appears not to be affected by treatment duration, disease progression or concomitant chemotherapy. This finding supports the application of trastuzumab at any stage of the disease.