Efficacy of Sym004 in Patients With Metastatic Colorectal Cancer With Acquired Resistance to Anti-EGFR Therapy and Molecularly Selected by Circulating Tumor DNA Analyses: A Phase 2 Randomized Clinical Trial.

IMPORTANCE: Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due to RAS and EGFR extracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR-refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. OBJECTIVE: To determine if continuous blockade of EGFR by Sym004 has survival benefit. DESIGN, SETTING, AND PARTICIPANTS: Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator's choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-type KRAS exon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. INTERVENTIONS: Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator's choice of treatment (arm C). MAIN OUTCOMES AND MEASURES: Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. RESULTS: A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n = 160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targeted EGFR ECD-mutated cancer cells, and a decrease in EGFR ECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients with EGFR ECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFR ECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). CONCLUSIONS AND RELEVANCE: Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. TRIAL REGISTRATION: clinicaltrialsregister.eu Identifier: 2013-003829-29.

Intracellular autofluorescence: a biomarker for epithelial cancer stem cells.

Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.

Micro segmented-flow in biochemical and cell-based assays.

Micro-segmented flow (e.g. in microfluidic channels, capillaries or a length of tubing) has become a promising technique in modern biology. Compared to conventional formats such as microtiter plates, sample volumes can be reduced about 1000-fold, thus allowing a massive reduction of assay costs and the use of samples available in low quantities, only (e.g. primary cells). Furthermore, assays can be highly parallelized and performed at superb spatio-temporal resolution. Here, we review the state-of-the-art in micro-segmented flow as applied in biochemical, cell- and multicellular organisms-based assays. We discuss likely future applications such as single cell / single organism proteomics and transcriptomics and point out the specific advantages and limitations compared to emulsion-based (droplet-based) approaches.

An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries.

Droplet-based microfluidic systems allow biological and chemical reactions to be performed on a drastically decreased scale. However, interfacing the outside world with such systems and generating high numbers of microdroplets of distinct chemical composition remain challenging. We describe here an automated system in which arrays of chemically distinct plugs are generated from microtiter plates. Each array can be split into multiple small-volume copies, thus allowing several screens of the same library. The system is fully compatible with further on-chip manipulation(s) and allows monitoring of individual plugs over time (e.g. for recording reaction kinetics). Hence the technology eliminates several bottlenecks of current droplet-based microfluidic systems and should open the way for (bio-)chemical and cell-based screens.

Coupling the inhibition of viral transduction with a positive fluorescence signal.

Cell-based assays for the inhibition of viral infections most commonly couple a positive signal (e.g., an increase in fluorescence) to the infection itself and not to its inhibition. When performing drug screens, compounds decreasing the signal are therefore considered as putative inhibitors. However, this approach can cause the selection of many false positives, since, for example, both killing of the host cell and inhibiting viral cell-entry results in the same signal. Using a model system based on murine leukemia virus (MLV) particles pseudotyped with the G-protein of vesicular stomatitis virus (VSV-G), we have developed generic assays coupling a positive readout to the inhibition of viral transduction. Consequently, the system favors drug candidates (and concentrations thereof) that do not harm human cells and significantly decreases the probability for selecting false positives. The assay allows Z-factors of approximately 0.9, takes cytotoxic side effects into account and could in theory be adapted for high-throughput screening of inhibitors against further viral species.

Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms.

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.

Determination of sulfonamides in milk after precolumn derivatisation by micellar liquid chromatography.

A simple method to identify and determine six sulfonamides (sodium sulfacetamide, sulfamethizole, sulfaguanidine, sulfamerazine, sulfathiazole and sulfamethoxazole) in milk by micellar liquid chromatography (MLC) is reported. The assay makes use of a precolumn diazotisation-coupling derivatisation including the formation of an azo dye that can be detected at 490 nm. Furthermore, the use of MLC as an analytical tool allows the direct injection of non-purified samples. The separation was performed with an 80 mM SDS-8.5% propanol eluent at pH 7. Analysis times are below 16 min with a complete resolution. Linearities (r>0.9999), as well as intra- and inter-day precision (below 2.7%), were studied in the validation of the method. The limits of detection and quantification ranged from approximately 0.72 to 0.94 and 2.4 to 3.1 ng mL(-1), respectively. The detection limit was below the maximum residue limit established by the European Community. Finally, recoveries in spiked milk samples were in the 83-103% range.