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Abstract Background During targeted treatment, HER2-positive breast cancers invariably lose HER2 DNA amplification. In contrast, and interestingly, HER2 proteins may be either lost or gained. To longitudinally and systematically appreciate complex/discordant changes in HER2 DNA/protein stoichiometry, HER2 DNA copy numbers and soluble blood proteins (aHER2/sHER2) were tested in parallel, non-invasively (by liquid biopsy), and in two-dimensions, hence HER2-2D. Methods aHER2 and sHER2 were assessed by digital PCR and ELISA before and after standard-of-care treatment of advanced HER2-positive breast cancer patients (n=37) with the antibody-drug conjugate (ADC) Trastuzumab-emtansine (T-DM1). Results As expected, aHER2 was invariably suppressed by T-DM1, but this loss was surprisingly mirrored by sHER2 gain, sometimes of considerable entity, in most (30/37; 81%) patients. This unorthodox split in HER2 oncogenic dosage was supported by reciprocal aHER2/sHER2 kinetics in two representative cases, and an immunohistochemistry-high status despite copy-number-neutrality in 4/5 available post-T-DM1 tumor re-biopsies from sHER2-gain patients. Moreover, sHER2 was preferentially released by dying breast cancer cell lines treated in vitro by T-DM1. Finally, sHER2 gain was associated with a longer PFS than sHER2 loss (mean PFS 282 vs 133 days, 95% CI [210-354] vs [56-209], log-rank test p=0.047), particularly when cases (n=11) developing circulating HER2-bypass alterations during T-DM1 treatment were excluded (mean PFS 349 vs 139 days, 95% CI [255-444] vs [45-232], log-rank test p=0.009). Conclusions HER2 gain is adaptively selected in tumor tissues and recapitulated in blood by sHER2 gain. Possibly, an increased oncogenic dosage is beneficial to the tumor during anti-HER2 treatment with naked antibodies, but favorable to the host during treatment with a strongly cytotoxic ADC such as T-DM1. In the latter case, HER2-gain tumors may be kept transiently in check until alternative oncogenic drivers, revealed by liquid biopsy, bypass HER2. Whichever the interpretation, HER2-2D might help to tailor/prioritize anti-HER2 treatments, particularly ADCs active on aHER2-low/sHER2-low tumors. Trial registration NCT05735392 retrospectively registered on January 31, 2023 https://www.clinicaltrials.gov/search?term=NCT05735392

This study presents the development and characterization of a disposable biochip for the detection of antibodies against the SARS-CoV-2 spike protein, a well-known target for vaccine and therapeutic development. This biochip is based on a one-dimensional photonic crystal (1DPC) deposited on a plastic substrate and designed to sustain Bloch surface waves (BSW) in the visible range. The experimental phase was carried out using the biochip in conjunction with a custom-made optical read-out platform capable of real-time refractometric detection and fluorescence-based end-point measurements. Our biochip was functionalized by immobilizing the receptor-binding domain of the spike protein onto the surface using a silanization process. Human serum samples, including a negative control and a positive sample from a recovered COVID-19 patient, were tested on the biochip. The experimental results show that the biochip discriminates between positive and negative samples in a label-free refractometric mode down to a 1:10 dilution of the sera and in quantum dot amplified refractometric and fluorescence mode down to 1:100 dilution. The results demonstrate the potential of the disposable biochip for sensitive and specific detection of COVID-19 antibodies.

In the present communication, we report on the exploitation of a Bloch surface wave-enhanced fluorescence scheme for the detection of Interleukin (IL)-10 in a protein-rich buffer mimicking a biological sample. IL-10 is a cytokine known for its potent anti-inflammatory and immunosuppressive effects. It is considered a valuable biomarker for prognostic prediction for both solid tumors and hematological malignancies, and recently, a distinguishing feature of hyperinflammation during severe viral infections. To demonstrate the validity of the technique, we transferred all the reagents and working concentrations used in a gold-standard technique, such as ELISA, to our assay, with a substantial reduction in the execution time and without using any enzymatic amplification during IL-10 recognition. We estimate a limit of detection (LoD) in terms of the concentration of IL-10 in solution of the order of 110 pg/mL (5.8 pM) with a 14% accuracy; in other terms, the presented technique is compatible with the assay range and resolution (1.6 pM) of commercial gold-standard ELISA kits. Moreover, such LoD successfully matches the concentrations reported in literature for IL-10 detection in COVID-19 patients, making the BSW-based sensors a viable solution for rapid and accurate screening of COVID-19-related molecules.

A one-dimensional photonic crystal (1DPC) based on a planar stack of dielectric layers is used as an optical transducer for biosensing, upon the coupling of TE-polarized Bloch Surface Waves (BSW). The structure is tailored with a polymeric layer providing a chemical functionality facilitating the covalent binding of orienting proteins needed for a subsequent grafting of antibodies in an immunoassay detection scheme. The polymeric layer is impregnated with Cy3 dye, in such a way that the photonic structure can exhibit an emissive behavior. The BSW-coupled fluorescence shift is used as a means for detecting refractive index variations occurring at the 1DPC surface, according to a label-free concept. The proposed working principle is successfully demonstrated in real-time tracking of protein G covalent binding on the 1DPC surface within a fluidic cell.

Optical biosensors based on one-dimensional photonic crystals sustaining Bloch surface waves are proposed to study antibody interactions and perform affinity studies. The presented approach utilizes two types of different antibodies anchored at the sensitive area of a photonic crystal-based biosensor. Such a strategy allows for creating two or more on-chip regions with different biochemical features as well as studying the binding kinetics of biomolecules in real time. In particular, the proposed detection system shows an estimated limit of detection for the target antibody (anti-human IgG) smaller than 0.19 nM (28 ng/mL), corresponding to a minimum surface mass coverage of 10.3 ng/cm2. Moreover, from the binding curves we successfully derived the equilibrium association and dissociation constants (KA = 7.5 × 107 M−1; KD = 13.26 nM) of the human IgG–anti-human IgG interaction.

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of the cancer biomarker ERBB2 in cell lysates. Overexpression of the ERBB2 protein is associated with aggressive breast cancer subtypes. To detect soluble ERBB2, we developed an optical set-up which operates in both label-free and fluorescence modes. The detection approach makes use of a sandwich assay, in which the one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies, in order to guarantee high specificity during the biological recognition. We present the results of exemplary protein G based label-free assays in complex biological matrices, reaching an estimated limit of detection of 0.5 ng/mL. On-chip and chip-to-chip variability of the results is addressed too, providing repeatability rates. Moreover, results on fluorescence operation demonstrate the capability to perform high sensitive cancer biomarker assays reaching a resolution of 0.6 ng/mL, without protein G assistance. The resolution obtained in both modes meets international guidelines and recommendations (15 ng/mL) for ERBB2 quantification assays, providing an alternative tool to phenotype and diagnose molecular cancer subtypes.