Marlene Thomas

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (89Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of 89Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of 89Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg 89Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent 89Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of 89Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of 89Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, 89Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.

Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues

Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin‐fixed paraffin‐embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin‐fixed paraffin‐embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery.

First-in-Human Phase I Study of Lumretuzumab, a Glycoengineered Humanized Anti-HER3 Monoclonal Antibody, in Patients with Metastatic or Advanced HER3-Positive Solid Tumors.

Purpose: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer. Experimental Design: Twenty-five patients with histologically confirmed HER3-expressing tumors received lumretuzumab (100, 200, 400, 800, 1,600, and 2,000 mg) every two weeks (q2w) in 3+3 dose-escalation phase. In addition, 22 patients were enrolled into an extension cohort at 2,000 mg q2w. Results: There were no dose-limiting toxicities. Common adverse events (any grade) included diarrhea (22 patients, 46.8%), fatigue (21 patients, 44.7%), decreased appetite (15 patients, 31.9%), infusion-related reactions (13 patients, 27.7%), and constipation (10 patients, 21.3%). The peak concentration (Cmax) and area under the concentration–time curve up to the last measurable concentration (AUClast) of lumretuzumab increased more than dose proportionally from 100 mg up to 400 mg. Linear PK was observed with doses ≥400 mg q2w indicating target-mediated drug disposition saturation. Downregulation of HER3 membranous protein was observed in on-treatment tumor biopsies from 200 mg, and was maximal at and above 400 mg. An ex vivo assay demonstrated increased activation potential of peripheral NK lymphocytes with lumretuzumab compared with a non-glycoengineered anti-HER3 antibody. Ten patients (21.3%) had stable disease and remained on study at a median of 111 days (range, 80–225 days). Conclusions: Lumretuzumab was well tolerated and showed evidence of clinical activity. Linear serum PK properties and plateauing of PD effects in serial tumor biopsies indicate optimal biologically active doses of lumretuzumab from 400 mg onwards. Clin Cancer Res; 22(4); 877–85. ©2015 AACR.

An optimized workflow for improved gene expression profiling for formalin-fixed, paraffin-embedded tumor samples

BackgroundWhole genome microarray gene expression profiling is the ‘gold standard’ for the discovery of prognostic and predictive genetic markers for human cancers. However, suitable research material is lacking as most diagnostic samples are preserved as formalin-fixed, paraffin-embedded tissue (FFPET). We tested a new workflow and data analysis method optimized for use with FFPET samples.MethodsSixteen breast tumor samples were split into matched pairs and preserved as FFPET or fresh-frozen (FF). Total RNA was extracted and tested for yield and purity. RNA from FFPET samples was amplified using three different commercially available kits in parallel, and hybridized to Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays. The array probe set was optimized in silico to exclude misdesigned and misannotated probes.ResultsFFPET samples processed using the WT-Ovation™ FFPE System V2 (NuGEN) provided 80% specificity and 97% sensitivity compared with FF samples (assuming values of 100%). In addition, in silico probe set redesign improved sequence detection sensitivity and, thus, may rescue potentially significant small-magnitude gene expression changes that could otherwise be diluted by the overall probe set background.ConclusionIn conclusion, our FFPET-optimized workflow enables the detection of more genes than previous, nonoptimized approaches, opening new possibilities for the discovery, validation, and clinical application of mRNA biomarkers in human diseases.

Phase Ib study of lumretuzumab plus cetuximab or erlotinib in solid tumor patients and evaluation of HER3 and heregulin as potential biomarkers of clinical activity

Purpose: This study investigated the safety, clinical activity, and target-associated biomarkers of lumretuzumab, a humanized, glycoengineered, anti-HER3 monoclonal antibody (mAb), in combination with the EGFR-blocking agents erlotinib or cetuximab in patients with advanced HER3-positive carcinomas. Experimental Design: The study included two parts: dose escalation and dose extension phases with lumretuzumab in combination with either cetuximab or erlotinib, respectively. In both parts, patients received lumretuzumab doses from 400 to 2,000 mg plus cetuximab or erlotinib according to standard posology, respectively. The effect of HRG mRNA and HER3 mRNA and protein expression were investigated in a dedicated extension cohort of squamous non–small cell lung cancer (sqNSCLC) patients treated with lumretuzumab and erlotinib. Results: Altogether, 120 patients were treated. One dose-limiting toxicity (DLT) in the cetuximab part and two DLTs in the erlotinib part were reported. The most frequent adverse events were gastrointestinal and skin toxicities, which were manageable. The objective response rate (ORR) was 6.1% in the cetuximab part and 4.2% in the erlotinib part. In the sqNSCLC extension cohort of the erlotinib part, higher tumor HRG and HER3 mRNA levels were associated with a numerically higher disease control rate but not ORR. Conclusions: The toxicity profile of lumretuzumab in combination with cetuximab and erlotinib was manageable, but only modest clinical activity was observed across tumor types. In the sqNSCLC cohort, there was no evidence of meaningful clinical benefit despite enriching for tumors with higher HRG mRNA expression levels. Clin Cancer Res; 23(18); 5406–15. ©2017 AACR.

Zr-89-Lumretuzumab PET Imaging before and during HER3 Antibody Lumretuzumab Treatment in Patients with Solid Tumors

Purpose: We evaluated biodistribution and tumor targeting of 89Zr-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)–targeting monoclonal antibody. Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received 89Zr-lumretuzumab and underwent positron emission tomography (PET). In part A, 89Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV). Results: Optimal PET conditions were found to be 4 and 7 days after administration of 89Zr-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4 (±1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis (n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (±8.2), 10.0% (±16.5), and 24.6% (±20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab. Conclusions: PET imaging showed biodistribution and tumor-specific 89Zr-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased 89Zr-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. Clin Cancer Res; 23(20); 6128–37. ©2017 AACR.

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.

Profiling of cMET and HER Family Receptor Expression in Pancreatic Ductal Adenocarcinomas and Corresponding Lymph Node Metastasis to Assess Relevant Pathways for Targeted Therapies: Looking at the Soil Before Planting the Seed.

Objectives Comprehensive assessment of cMET and HER family receptor tyrosine kinases expression, changes of expression during metastatic progression, amplification status of the MET gene, and correlations with patient characteristics in pancreatic ductal adenocarcinoma (PDAC) was conducted. Methods We investigated 56 PDACs and corresponding lymph node metastases for HER1 to HER4 and cMET expression by immunohistochemistry, as well as cMET gene copy numbers by chromogenic in situ hybridization. Results Of all receptor tyrosine kinases evaluated, cMET expression was highest with 46.5% of tumors showing moderate or strong expression and a weak correlation with gene copy number status (P = 0.04; Spearman &rgr; = 0.28). cMET expression was increased in metastases. In contrast, expression levels of HER family receptors were generally low both in primaries and metastases. A weak yet significant correlation of HER1 and cMET expression levels was observed (P < 0.001; Spearman &rgr; = 0.44) and HER1 was often present in poorly differentiated tumors (G3, P = 0.049). Conclusions Our data suggest that cMET might constitute an interesting molecule for combining targeted and chemotherapeutic approaches in PDAC, because expression is frequent and increased during metastatic progression. In PDAC, cMET protein expression might be a more useful stratification biomarker than cMET gene amplification, which does not seem to be its primary regulator.

Detection of Truncated HER2 Forms in Formalin-Fixed, Paraffin-Embedded Breast Cancer Tissue Captures Heterogeneity and Is Not Affected by HER2-Targeted Therapy

Truncated forms of HER2, previously identified in subsets of HER2-positive breast cancer, originate from proteolytic extracellular domain (ECD) cleavage or alternative translation initiation. They lack ECD but may retain intracellular domain functionality, potentially associated with unfavorable prognosis, metastasis, and decreased sensitivity to antibody-based HER2-targeted therapy. To study the distribution of truncated HER2 in breast cancer, we detected loss of membrane-bound ECD independently of its molecular origin in paraffin sections, combining multispectral unmixing of chromogenic duplex IHC for HER2 ECD and intracellular domain with advanced image analysis. HER2 C-terminal fragment 611-transfected MCF7 and 4-aminophenylmercuric acetate-treated SKBR3 cell lines were used as controls. Applying a prototype work flow to whole sections, paired surgical resection/core needle biopsy samples, and paired samples from 69 patients of a phase 2 neoadjuvant clinical trial, we observed unexpected heterogeneity of ECD loss at the single-cell level, and in different areas of individual tumors, indicating that extent and localization of HER2 ECD loss add relevant information to averaging truncated HER2 across whole sections. We show acceptable run-to-run variation (coefficient of variation, <0.15), image analysis results in moderate agreement with conventional slide assessment (Cohens κ = 0.59), and no obvious interference with previous HER2-ECD-targeted therapy. We conclude that duplex IHC and digital image processing extend current approaches of truncated HER2 detection.

Die Bedeutung von Biobanken für die klinische Entwicklung

Access to samples in biobanks and collection of samples for evaluation of biomarkers in clinical trials are an essential basis for the identification and development of biomarkers. From the perspective of a research-based pharmaceutical company identification of biomarkers and the accompanying diagnostics are an essential prerequisite for the further evolution of personalised healthcare-and the key to more effective and efficient healthcare. Research-based pharmaceutical companies can basically use four types of biobanks: biobanks of university hospitals, commercial providers, collaborative groups and company-owned biobanks. Areas of application, arising from the use of biobanks in the context of clinical development, are collection of prevalence data, evaluation of biomarker stability in different disease stages, technical validation of assays, an optimized course of clinical studies by focusing on defined, biomarker-stratified groups of patients and pharmacogenetic research. Challenges are, in particular, the availability of clinically annotated samples and tissue matching blood samples, in addition to sample quality, number and amount. An acceptable legal and regulatory framework, as well as the positive perception of biomarker data by politicians and the public, are important prerequisites for translational research for identification of biomarkers in clinical studies. Also, the early establishment of research alliances between academia and the pharmaceutical industry are required to transfer research results in new strategies for prevention, diagnosis and treatment of patients.