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Purpose: Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand-receptor pair frequently overexpressed in solid tumors. TWEAK:Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFkB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1k monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14.Experimental Design: Dose escalations, over a 200-to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of TWEAK:Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics.Results: In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t 1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment.Conclusion: RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced TWEAK:Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258-66. Ó2014 AACR.

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

In our study we systematically compared the alternative fixatives acidified formal alcohol (AFA), PAXgene®, HOPE®, and combinations of AFA or formalin with ultrasound treatment to standard (buffered) formalin fixation. We examined general morphology and detectability of protein structures by immunohistochemistry of the membrane receptors epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), and phosphorylated human epidermal growth factor receptor 2 (phospho-HER2). In order to allow for stringent comparability of different fixation techniques, we used matched mouse xenograft tumor samples from three different human cancer cell lines (colon, ovarian, and non-small cell lung cancer), either fixed conventionally with formalin or an alternative fixative. Tissue morphology after fixation with AFA and PAXgene® was comparable to formalin-fixed paraffin-embedded tissue (FFPET) morphology. Ultrasound fixations resulted in slightly inferior morphology and HOPE® fixation preserved morphology only poorly compared to FFPET in this system. None of the tested alternative fixatives enabled immunohistochemical detectability of all three targets in the same manner as FFPET. Pronounced staining was possible for EGFR and IGF-1R with all alternative fixatives but HOPE®, and phospho-HER2 staining was only noteworthy with formalin-ultrasound-fixed tissue. Therefore, the use of alternative fixatives comes with the need for careful validation of obtained IHC results individually for each target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-012-1248-5) contains supplementary material, which is available to authorized users.

Introduction:CD38 is a type II transmembrane glycoprotein widely expressed in many hematological malignancies including multiple myeloma (MM). MOR202, a HuCAL-derived, human IgG1 CD38 monoclonal antibody, induces antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). MOR202 does not induce complement-dependent cytotoxicity, which is suspected to be a major contributor to infusion-related reactions (IRRs). Preclinical models of MM demonstrate high single-agent antitumor activity of MOR202 and synergy in combination with immunomodulatory drugs (IMiDs), lenalidomide (LEN) or pomalidomide (POM). Methods: This is an interim analysis of a multicenter, dose-escalation phase I/IIa study of MOR202 in relapsed or refractory (RR)MM. Preliminary safety and efficacy data from 3 patient cohorts treated with clinically relevant doses of MOR202 (administered as an IV 2-hour infusion), alone or in combination with an IMiD are presented: MOR202 4, 8 and 16 mg/kg weekly; MOR202 8 or 16 mg/kg weekly with either LEN or POM. All patients in these cohorts also received low dose dexamethasone. Primary objectives were to evaluate the safety, maximum tolerated dose (MTD) and recommended phase II dose of MOR202. Secondary objectives included an assessment of overall response rate, duration of response and progression-free survival. Results: As of July 12, 2016, a total of 66 patients had been treated; 31 in clinically relevant cohorts, including 18 patients receiving MOR202 alone, 8 receiving MOR202 + LEN and 5 receiving MOR202 + POM. Patients treated with MOR202 alone and MOR202 + POM had both received a median of 4 prior lines of therapy; 78% and 100% had been refractory to last prior treatment, respectively. Patients treated with MOR202 + LEN had received a median of 2 prior lines of therapy and 50% had been refractory to last prior treatment. Most of the patients had received bortezomib, LEN, cyclophosphamide, and melphalan alone or in combination with autologous stem cell transplant as part of their prior regimens. In this trial the MTD has not been reached yet. MOR202 alone or in combination with an IMiD was well tolerated, with mainly hematological toxicity reported. A 2-hour MOR202 infusion was feasible in all patients. In the clinically relevant cohorts only 1 patient discontinued due to an adverse event considered to be related to MOR202 (platelet count decreased) and no deaths related to any of the study drugs occurred. IRRs were seen in only 3/31 (10%) patients, all occurring during the first infusion. All IRRS were ≤ grade 2. So far, 28 patients were evaluable for response in the MOR202 clinically relevant cohorts. Of 16 evaluable patients in the MOR202 alone cohort, 3 partial responses (19%) and 2 very good partial responses (13%) were reported. In the MOR202 + LEN cohort 5/7 partial responses were seen, and 3/5 patients responded to MOR202 + POM treatment including 2 complete responses. Median time to response was 4 weeks, with responses tending to deepen over time. Most responses (10/13) are ongoing with the longest duration of response currently being 48 weeks. Preliminary analysis in 5 patients revealed preservation of high CD38 levels on MM cells under MOR202 therapy, with a mean decrease of only 10% from baseline to day 1 cycle 2 (4 weeks). Conclusions: In this analysis, a 2-hour infusion of MOR202 (up to 16 mg/kg) alone, or in combination with POM or LEN showed a very good safety profile, particularly an excellent infusion tolerability in heavily pretreated patients with RRMM. Promising preliminary efficacy and long-lasting tumor control was seen for MOR202 +/- IMiDs. The data suggest that CD38 expression on patient MM cells is preserved during treatment. Disclosures Raab: Novartis: Consultancy, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Amgen: Consultancy, Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Agis:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Einsele:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Engelhardt:Amgen: Research Funding; Janssen: Research Funding; MSD: Research Funding; Celgene: Research Funding. Ferstl:Novartis: Other: Case report presentation; Bristol Myers Squibb: Other: Advisory Board. Weisel:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Onyx: Consultancy; BMS: Consultancy, Honoraria; Novartis: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Jarutat:MorphoSys AG: Employment. Weinelt:MorphoSys: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Peschel:MophoSys: Honoraria.

Determination of protein phosphorylation using routinely collected surgical specimens results in artefacts which do not reflect a tumour's true states of pathway activation. Valid measurement of phosphorylated biomarkers requires that tissue collection procedures are tightly controlled, avoiding ischaemia and large-specimen fixation.

It has been shown that a repetitive motif with the sequence FKEL(F) within the Ki-67 antigen (pKi-67) serves as an epitope for the Ki-67 antibody and equivalent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in formalin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and characterization of new monoclonal Ki-67 equivalent antibodies in an in vitro approach. To select pKi-67 reactive phage antibodies, we used a large naive Fab-phage library (Human Combinatorial Antibody Library; HuCAL). We implemented a panning strategy against two different overlapping peptides, both containing the 'FKELF' epitope. ELISA screening of randomly picked phage antibody clones after the third selection round yielded six highly reactive clones against the 'FKELF' epitope, of which five were found to be reactive in FFPE tissue, showing a Ki-67 equivalent staining pattern. Substitutional epitope analysis on peptide arrays of the new recombinant pKi-67 binders and of the established murine clones Ki-67, Mib-1 and Mib-5 were carried out to compare their fine specificities. The results suggest that the lysine residue in the epitope is critical for recognition of Ki-67 antigen in FFPE tissue.

Purpose: The TWEAK-Fn14 pathway represents a novel anticancer target that is being actively investigated. Understanding the relationship between pharmacokinetics of anti-TWEAK therapeutics and tumor pharmacodynamics is critical. We investigated exposure-response relationships of RG7212, an anti-TWEAK mAb, in patients with Fn14-expressing tumors.Experimental Design: Patients with Fn14-positive tumors (IHC!1þ) treated in a phase I first-in-human study with ascending doses of RG7212 were the basis for this analysis. Pharmacokinetics of RG7212 and dynamics of TWEAK were determined, as were changes in tumor TWEAK-Fn14 signaling in paired pre-and posttreatment tumor biopsies. The objectives of the analysis were to define exposure-response relationships and the relationship between pretreatment tumor Fn14 expression and pharmacodynamic effect. Associations between changes in TWEAK-Fn14 signaling and clinical outcome were explored.Results: Thirty-six patients were included in the analysis. RG7212 reduced plasma TWEAK to undetectable levels at all observed RG7212 exposures. In contrast, reductions in tumor Fn14 and TRAF1 protein expression were observed only at higher exposure (!300 mg à h/mL). Significant reductions in tumor Ki-67 expression and early changes in serum concentrations of CCL-2 and MMP-9 were observed exclusively in patients with higher drug exposure who had high pretreatment tumor Fn14 expression. Pretreatment tumor Fn14 expression was not associated with outcome, but a trend toward longer time on study was observed with high versus low RG7212 exposure.Conclusions: RG7212 reduced tumor TWEAK-Fn14 signaling in a systemic exposure-dependent manner. In addition to higher exposure, relatively high Fn14 expression might be required for pharmacodynamic effect of anti-TWEAK monoclonal antibodies.