Direct competitive assay for HER2 detection in human plasma using Bloch surface wave-based biosensors.

The overexpression and/or amplification of the HER2/neu oncogene has been proposed as a prognostic marker in breast cancer. The detection of the related peptide HER2 remains a grand challenge in cancer diagnosis and for therapeutic decision-making. Here, we used a biosensing device based on Bloch Surface Waves excited on a one-dimensional photonic crystal (1DPC) as valid alternative to standard techniques. The 1DPC was optimized to operate in the visible spectrum and the biosensor optics has been designed to combine label-free and fluorescence operation modes. This feature enables a real-time monitoring of a direct competitive assay using detection mAbs conjugated with quantum dots for an accurate discrimination in fluorescence mode between HER2-positive/negative human plasma samples. Such a competitive assay was implemented using patterned alternating areas where HER2-Fc chimera and reference molecules were bio-conjugated and monitored in a multiplexed way. By combining Label-Free and fluorescence detection analysis, we were able to tune the parameters of the assay and provide an HER2 detection in human plasma in less than 20 min, allowing for a cost-effective assay and rapid turnaround time. The proposed approach offers a promising technique capable of performing combined label-free and fluorescence detection for both diagnosis and therapeutic monitoring of diseases.

The Molecular Tumor Board of the Regina Elena National Cancer Institute: from accrual to treatment in real-world.

BACKGROUND: Molecular Tumor Boards (MTB) operating in real-world have generated limited consensus on good practices for accrual, actionable alteration mapping, and outcome metrics. These topics are addressed herein in 124 MTB patients, all real-world accrued at progression, and lacking approved therapy options. METHODS: Actionable genomic alterations identified by tumor DNA (tDNA) and circulating tumor DNA (ctDNA) profiling were mapped by customized OncoKB criteria to reflect diagnostic/therapeutic indications as approved in Europe. Alterations were considered non-SoC when mapped at either OncoKB level 3, regardless of tDNA/ctDNA origin, or at OncoKB levels 1/2, provided they were undetectable in matched tDNA, and had not been exploited in previous therapy lines. RESULTS: Altogether, actionable alterations were detected in 54/124 (43.5%) MTB patients, but only in 39 cases (31%) were these alterations (25 from tDNA, 14 from ctDNA) actionable/unexploited, e.g. they had not resulted in the assignment of pre-MTB treatments. Interestingly, actionable and actionable/unexploited alterations both decreased (37.5% and 22.7% respectively) in a subset of 88 MTB patients profiled by tDNA-only, but increased considerably (77.7% and 66.7%) in 18 distinct patients undergoing combined tDNA/ctDNA testing, approaching the potential treatment opportunities (76.9%) in 147 treatment-naïve patients undergoing routine tDNA profiling for the first time. Non-SoC therapy was MTB-recommended to all 39 patients with actionable/unexploited alterations, but only 22 (56%) accessed the applicable drug, mainly due to clinical deterioration, lengthy drug-gathering procedures, and geographical distance from recruiting clinical trials. Partial response and stable disease were recorded in 8 and 7 of 19 evaluable patients, respectively. The time to progression (TTP) ratio (MTB-recommended treatment vs last pre-MTB treatment) exceeded the conventional Von Hoff 1.3 cut-off in 9/19 cases, high absolute TTP and Von Hoff values coinciding in 3 cases. Retrospectively, 8 patients receiving post-MTB treatment(s) as per physician's choice were noted to have a much longer overall survival from MTB accrual than 11 patients who had received no further treatment (35.09 vs 6.67 months, p = 0.006). CONCLUSIONS: MTB-recommended/non-SoC treatments are effective, including those assigned by ctDNA-only alterations. However, real-world MTBs may inadvertently recruit patients electively susceptible to diverse and/or multiple treatments.

Widespread in vivo efficacy of The-0504: A conditionally-activatable nanoferritin for tumor-agnostic targeting of CD71-expressing cancers.

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers). Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations. Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX. Expression increases up to 4-fold and is more persistent as compared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting.

Metastatic Melanoma: Liquid Biopsy as a New Precision Medicine Approach.

Precision medicine has driven a major change in the treatment of many forms of cancer. The discovery that each patient is different and each tumor mass has its own characteristics has shifted the focus of basic and clinical research to the singular individual. Liquid biopsy (LB), in this sense, presents new scenarios in personalized medicine through the study of molecules, factors, and tumor biomarkers in blood such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes and circulating tumor microRNAs (ct-miRNAs). Moreover, its easy application and complete absence of contraindications for the patient make this method applicable in a great many fields. Melanoma, given its highly heterogeneous characteristics, is a cancer form that could significantly benefit from the information linked to liquid biopsy, especially in the treatment management. In this review, we will focus our attention on the latest applications of liquid biopsy in metastatic melanoma and possible developments in the clinical setting.

Unusual phylogenetic tree and circulating actionable ESR1 mutations in an aggressive luminal/HER2-low breast cancer: Case report.

Under therapeutic pressure aggressive tumors evolve rapidly. Herein, a luminal B/HER2-low breast cancer was tracked for >3 years during a total of 6 largely unsuccessful therapy lines, from adjuvant to advanced settings. Targeted next generation sequencing (NGS) of the primary lesion, two metastases and 14 blood drawings suggested a striking, unprecedented coexistence of three evolution modes: punctuated, branched and convergent. Punctuated evolution of the trunk was supported by en bloc inheritance of a large set (19 distinct genes) of copy number alterations. Branched evolution was supported by the distribution of site-specific SNVs. Convergent evolution was characterized by a unique asynchronous expansion of three actionable (OncoKB level 3A) mutations at two consecutive ESR1 codons. Low or undetectable in all the sampled tumor tissues, ESR1 mutations expanded rapidly in blood during HER2/hormone double-blockade, and predicted life-threatening local progression at lung and liver metastatic foci. Dramatic clinical response to Fulvestrant (assigned off-label exclusively based on liquid biopsy) was associated with clearance of all 3 subclones and was in stark contrast to the poor therapeutic efficacy reported in large liquid biopsy-informed interventional trials. Altogether, deconvolution of the tumor phylogenetic tree, as shown herein, may help to customize treatment in breast cancers that rapidly develop refractoriness to multiple drugs.

A Distinctive microRNA (miRNA) Signature in the Blood of Colorectal Cancer (CRC) Patients at Surgery.

BACKGROUND: Liquid biopsy (LB) provides an examination of the peripheral blood of cancer patients for circulating tumor cells, cell-free nucleic acids and microRNAs (miRNAs) and is an established tool of precision medicine. Unlike most previous LB studies that focused on advanced metastatic colorectal cancer (CRC), we assessed miRNA dysregulation in blood samples obtained on the day of surgery from patients with primary CRC lesions but no clinical evidence of extra-colonic diffusion. In this study, plasma preparation included miRNAs associated to exosomes, but excluded large macrovesicles from the preparation. METHODS: The miRNA profile in plasma isolated from a cohort of 35 CRC patients at the day of surgery was analyzed by Next Generation Sequencing (NGS) and further confirmed by droplet digital RT-PCR (dd-RT-PCR). RESULTS: A miR-141-3p/miR-221-3p/miR-222-3p upregulation signature previously described in advanced CRC did not discriminate the analyzed early-CRC cohort from six tumor-free donors (Tf-D). In contrast, NGS-based miRNome analysis of a training cohort of five CRC and three tumor-free donors identified a novel, distinct nine miRNA signature comprising five up-regulated and four down-regulated miRNAs, six of which could be confirmed in the full CRC and tumor-free donor validation dataset by dd-RT-PCR. Additionally, a KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutant status was correlated with the plasma content of three identified miRNAs. CONCLUSIONS: When the data obtained were comparatively evaluated, at least one of the miRNAs belonging to the signature list was found to be dysregulated in 34/35 (97.1%) of our early-CRC plasma samples. The miRNA list provides diagnostic markers as well as possible molecular targets for protocols focusing on "microRNA therapeutics".

Cross-sectional analysis of circulating tumor DNA in primary colorectal cancer at surgery and during post-surgery follow-up by liquid biopsy.

BACKGROUND: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated. METHODS: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls. RESULTS: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively. CONCLUSIONS: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.

Bioassay engineering: a combined label-free and fluorescence approach to optimize HER2 detection in complex biological media.

We report on the combined label-free/fluorescence use of one-dimensional photonic crystals to optimize cancer biomarker detection in complex biological media. The optimization of the assay working parameters permits us to maximize the final response of the biosensor. The detection approach utilizes a sandwich assay, in which one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies in order to guarantee high specificity during biological recognition. The multiple outcomes generated by such optimization experiments permitted us to determine the effective capture efficiency and the repeatability of the immobilization process, which was estimated to be close to 5%. By exploiting the resolution of the fluorescence operation mode, we studied non-specific interactions in different blocking agents, different analyte diluting buffers, and diverse concentrations of the detection antibody. As a clinically relevant biomarker, we selected the trans-membrane receptor tyrosine kinase HER2. HER2 regulates a variety of cell proliferation, growth, and differentiation pathways and its over-expression occurs in approximately 20-30% of breast cancer worldwide. As a final application, we transferred all the optimized working parameters to HER2 cancer biomarker assays in a complex biological environment. The label-free and fluorescence results obtained by analyzing MCF-7 (HER2 low positive) and 32D (HER2 negative) cell lysates demonstrate that we can successfully discriminate the two lysates.