Frank Stenner-Liewen

CADD, a Chlamydia Protein That Interacts with Death Receptors

We report here the identification of a bacterial protein capable of interacting with mammalian death receptors in vitro and in vivo. The protein is encoded in the genome of Chlamydia trachomatis and has homologues in otherChlamydia species. This protein, which we refer to as “ C hlamydia protein associating with death domains” (CADD), induces apoptosis in a variety of mammalian cell lines when expressed by transient gene transfection. Apoptosis induction can be blocked by Caspase inhibitors, indicating that CADD triggers cell death by engaging the host apoptotic machinery. CADD interacts with death domains of tumor necrosis factor (TNF) family receptors TNFR1, Fas, DR4, and DR5 but not with the respective downstream adaptors. In infected epithelial cells, CADD is expressed late in the infectious cycle of C. trachomatisand co-localizes with Fas in the proximity of the inclusion body. The results suggest a role for CADD modulating the apoptosis pathways of cells infected, revealing a new mechanism of host-pathogen interaction.

Rational development of high-affinity T-cell receptor-like antibodies

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

Expression and potential role of Fas-associated phosphatase-1 in ovarian cancer.

Fas-associated phosphatase-1 (FAP-1) is a protein-tyrosine phosphatase that binds the cytosolic tail of Fas (Apo1, CD95), presumably regulating Fas-induced apoptosis. Elevations of FAP-1 protein levels in some tumor cell lines have been correlated with resistance to Fas-induced apoptosis. To explore the expression of FAP-1 in ovarian cancer cell lines and archival tumor specimens, mouse monoclonal and rabbit polyclonal antibodies were generated against a FAP-1 peptide and recombinant FAP-1 protein. These antibodies were used for immunoblotting, immunohistochemistry, and flow-cytometry analysis of FAP-1 expression in the Fas-sensitive ovarian cancer lines HEY and BG-1, and in the Fas-resistant lines OVCAR-3 FR and SK-OV-3. All methods demonstrated high levels of FAP-1 in the resistant lines OVCAR-3 FR and SK-OV-3, but not in the Fas-sensitive lines HEY and BG-1. Furthermore, levels of FAP-1 protein also correlated with the amounts of FAP-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction analysis. FAP-1 protein levels were investigated by immunoblotting in the National Cancer Institutes panel of 60 human tumor cell lines. Although FAP-1 failed to correlate with Fas-resistance across the entire tumor panel, Fas-resistance correlated significantly with FAP-1 expression (P: < or = 0.05) and a low Fas/FAP-1 ratio (P: < or = 0.028) in ovarian cancer cell lines. FAP-1 expression was also evaluated in 95 archival ovarian cancer specimens using tissue-microarray technology. FAP-1 was expressed in nearly all tumors, regardless of histological type or grade, stage, patient age, response to chemotherapy, or patient survival. We conclude that FAP-1 correlates significantly with Fas resistance in ovarian cancer cell lines and is commonly expressed in ovarian cancers.

BAG1 over-expression in brain protects against stroke.

The co‐chaperone BAG1 binds and regulates 70 kDa heat shock proteins (Hsp70/Hsc70) and exhibits cytoprotective activity in cell culture models. Recently, we observed that BAG1 expression is induced during neuronal differentiation in the developing brain. However, the in vivo effects of BAG1 during development and after maturation of the central nervous system have never been examined. We generated transgenic mice over‐expressing BAG1 in neurons. While brain development was essentially normal, cultured cortical neurons from transgenic animals exhibited resistance to glutamate‐induced, apoptotic neuronal death. Moreover, in an in vivo stroke model involving transient middle cerebral artery occlusion, BAG1 transgenic mice demonstrated decreased mortality and substantially reduced infarct volumes compared to wild‐type littermates. Interestingly, brain tissue from BAG1 transgenic mice contained higher levels of neuroprotective Hsp70/Hsc70 protein but not mRNA, suggesting a potential mechanism whereby BAG1 exerts its anti‐apoptotic effects. In summary, BAG1 displays potent neuroprotective activity in vivo against stroke, and therefore represents an interesting target for developing new therapeutic strategies including gene therapy and small‐molecule drugs for reducing brain injury during cerebral ischemia and neurodegenerative diseases.

Infectious Port Complications Are More Frequent in Younger Patients with Hematologic Malignancies than in Solid Tumor Patients

Background: We assessed longevity and complications of totally implantable venous access devices in oncology patients. Methods: 197 patients received a total of 201 port devices via the subclavian vein for delivery of chemotherapy between January 1, 2005, and December 31, 2006. We reviewed the patient charts for port-related complications and risk factors until July 31, 2007. Results:A total of 47,781 catheter days were analyzed (median, 175 days; range, 1–831). Forty-six different complications occurred (0.96 complications/1,000 catheter days). The only risk factor significantly associated with a higher complication rate was younger age. Older patients had a lower risk for developing complications with a risk reduction of 2.4% for each year. There were no differences regarding underlying tumor, gender, access side, method of placement (subclavian/cephalic vein) or implanting team (thoracic versus visceral surgery). A trend was seen for shorter port longevity in hematologic patients compared to oncologic patients (p = 0.059). The former developed significantly more port-associated infections than solid tumor patients [11/53 cases (21%) versus 2/148 cases (1.4%); p < 0.0001]. Conclusions: Port-associated infections were mostly observed in younger patients with hematologic neoplasms. Prospective trials should be performed to evaluate the benefit of a prophylactic antimicrobial lock in these selected patients.

Daily Pomegranate Intake Has No Impact on PSA Levels in Patients with Advanced Prostate Cancer - Results of a Phase IIb Randomized Controlled Trial.

Pomegranate has been shown to prolong PSA doubling time in early prostate cancer, but no data from a placebo controlled trial has been published yet. The objective of this study was to prospectively evaluate the impact of pomegranate juice in patients with prostate cancer. We conducted a phase IIb, double blinded, randomized placebo controlled trial in patients with histologically confirmed prostate cancer. Only patients with a PSA value ≥ 5ng/ml were included. The subjects consumed 500 ml of pomegranate juice or 500 ml of placebo beverage every day for a 4 week period. Thereafter, all patients received 250 ml of the pomegranate juice daily for another 4 weeks. PSA values were taken at baseline, day 14, 28 and on day 56. The primary endpoint was the detection of a significant difference in PSA serum levels between the groups after one month of treatment. Pain scores and adherence to intervention were recorded using patient diaries. 102 patients were enrolled. The majority of patients had castration resistant prostate cancer (68%). 98 received either pomegranate juice or placebo between October 2008 and May 2011. Adherence to protocol was good, with 94 patients (96%) completing the first period and 87 patients (89%) completing both periods. No grade 3 or higher toxicities occurred within the study. No differences were detected between the two groups with regard to PSA kinetics and pain scores. Consumption of pomegranate juice as an adjunct intervention in men with advanced prostate cancer does not result in significant PSA declines compared to placebo.

Distinct genetic evolution patterns of relapsing diffuse large B-cell lymphoma revealed by genome-wide copy number aberration and targeted sequencing analysis.

Recurrences of diffuse large B-cell lymphomas (DLBCL) result in significant morbidity and mortality, but their underlying genetic and biological mechanisms are unclear. Clonal relationship in DLBCL relapses so far is mostly addressed by the investigation of immunoglobulin (IG) rearrangements, therefore, lacking deeper insights into genome-wide lymphoma evolution. We studied mutations and copy number aberrations in 20 paired relapsing and 20 non-relapsing DLBCL cases aiming to test the clonal relationship between primaries and relapses to track tumors’ genetic evolution and to investigate the genetic background of DLBCL recurrence. Three clonally unrelated DLBCL relapses were identified (15%). Also, two distinct patterns of genetic evolution in clonally related relapses were detected as follows: (1) early-divergent/branching evolution from a common progenitor in 6 patients (30%), and (2) late-divergent/linear progression of relapses in 11 patients (65%). Analysis of recurrent genetic events identified potential early drivers of lymphomagenesis (KMT2D, MYD88, CD79B and PIM1). The most frequent relapse-specific events were additional mutations in KMT2D and alterations of MEF2B. SOCS1 mutations were exclusive to non-relapsing DLBCL, whereas primaries of relapsing DLBCL more commonly displayed gains of 10p15.3–p12.1 containing the potential oncogenes PRKCQ, GATA3, MLLT10 and ABI1. Altogether, our study expands the knowledge on clonal relationship, genetic evolution and mutational basis of DLBCL relapses.

Concomitant statin use does not impair the clinical outcome of patients with diffuse large B cell lymphoma treated with rituximab-CHOP

Preclinical data indicated a detrimental effect of statins on the anti-lymphoma activity of rituximab. We evaluated the impact of concomitant statin medication on the response and survival of patients with diffuse large B cell lymphoma (DLBCL) receiving rituximab–cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) as first-line therapy. Medical histories of patients with DLBCL who were treated with R-CHOP as first-line therapy were assessed for concomitant statin use, response after completion of chemotherapy, event-free survival (EFS), and overall survival (OS). Furthermore, 2-[18F]fluor-2-deoxyglucose (FDG)-PET/CT results after completion of first-line therapy were compared between the groups. Overall, 145 patients with DLBCL treated with R-CHOP from January 2001 to December 2009 were analyzed. Twenty-one (15%) patients received statins throughout therapy. Five-year EFS was 67.3% in patients without statins compared with 79% in patients receiving statins during R-CHOP (HR, 0.47; 95% CI, 0.15–1.54, p = 0.2). Five-year OS was 81.4% for patients without statins compared with 93.3% for patients taking statins (HR, 0.58; 95% CI 0.07–4.55, p = 0.6). There were no statistically significant differences in the rates of complete remissions between the two groups (75% in the non-statin group versus 86% in the statin group, p = 0.45). A trend toward a lower rate of complete metabolic responses in FDG-PET/CT after chemotherapy was seen in patients without statin medication compared with the patients taking statins (84% versus 92%, p = 0.068). Concomitant statin use had no adverse impact on response to chemotherapy, EFS, and OS in patients treated with R-CHOP for DLBCL.

Temsirolimus Is Highly Effective as Third-Line Treatment in Chromophobe Renal Cell Cancer

We report unexpectedly high efficacy of temsirolimus as third-line treatment in a patient with metastatic chromophobe renal cell carcinoma. After failure of two sequentially administered tyrosine kinase inhibitors, treatment with temsirolimus resulted in a prolonged partial remission of 14 months, and the response is still continuing. Up to now, no data from randomized clinical studies have been published addressing the question of efficacy of temsirolimus as third-line treatment after failure of tyrosine kinase inhibitors. The case presented here implies that temsirolimus could be a viable option for patients with metastatic chromophobe renal cell carcinoma.

Cerebral vasculitis mimicking intracranial metastatic progression of lung cancer during PD-1 blockade

BackgroundStimulation of the immune system by targeting the PD-1/PD-L1 pathway can result in activation of anti-tumor immunity. Besides its clinical benefit immune checkpoint therapy leads to significant immune-related adverse events (irAEs). Some rare irAEs are not well described yet but are critical in patient management.Case presentationHere, we describe a case of autoimmune cerebral vasculitis/encephalitis after PD-1 inhibitor treatment for metastatic adenocarcinoma of the lung. Upon PD-1 blockade, the patient developed cerebral lesions, while having disease stabilization of extracranial metastases. Imaging suggested that the patient had new progressing brain metastases. Despite stereotactic irradiation the lesions progressed further. The largest lesion became symptomatic and had to be surgically resected. On examination, cerebral vasculitis was detected but not evidence of metastatic lung cancer. Analysis of the patient’s serum revealed the presence of antinuclear antibodies that were already present before starting PD-1 blockade. In addition, we also found anti-vascular endothelial antibodies in the serum.ConclusionThis finding suggests that the patient had preformed autoantibodies and the checkpoint inhibitor induced a clinically relevant autoimmune disease. Taken together, encephalitic lesions in patients under PD-1/PD-L1 blockade can mimic metastatic brain lesions and this rare irAE has to be considered as a differential diagnosis in patients treated with immunotherapy.