Multiplex assays enable the measurement of multiple analytes in low input samples, including tumour biopsies and circulating tumour derived cells, exosomes, cell-free DNA (cfDNA) and other molecules. Molecular classification of primary tissue, tumour heterogeneity, metastatic tissue, and early detection of therapy resistance requires the development of sensitive and precise multiplex assays with a streamlined and robust workflow.
The Innoplex assay is an RNA bead-based multiplex assay that determines the expression of multiple genes, through direct analysis of lysed samples and signal amplification (no need for nucleic acid extraction and amplification). This method utilises the xMAP Luminex Technology and the Invitrogen QuantiGene Plex Assay (Thermo Fisher), combined with biomarker panels derived from research outputs. In this article, we describe the applications of innovative methodologies to classify tumours and study disease progression, utilising low abundance tissue samples, circulating cells and cellular components in blood.
Introduction
Molecular classification of tumours, their heterogeneity and propensity to metastasis are the leading cause of cancer-related death. Globally, cancer accounted for 9.6 million deaths in 2018, hence, innovative methods are an unmet clinical need to support early diagnosis, proper diagnosis of heterogenous disease and provide the tools for patient surveillance for early detection of disease progression (metastasis) and therapy resistance.
The innovative multiplex assays (Innoplex) are RNA bead-based assays that measure expression of multiple tumour associated biomarkers in low abundance samples. These methodologies were optimised for multiplex digitalised readout using various sample sources ranging from archival formalin fixed paraffin embedded tissues and blood derived exosomes. In this article, we summarise the Innoplex assays based on the xMAP Luminex Technology and the Invitrogen QuantiGene Plex Assay, the research outputs from the University of Malta in terms of the biomarker panels and the commercialisation of the assays through Omnigene Medical Technologies Ltd.
Workflow of the innovative molecular profiling technology
The Innoplex multiplex assays are based on the integration of the Invitrogen QuantiGene Plex Assay (Thermo Fisher Scientific) and the xMAP Luminex technology enabling multiplexing of the technique, and on novel panel of biomarkers developed by the Laboratory of Molecular Oncology at the University of Malta, headed by Professor Godfrey Grech. The technologies and the research output provide the versatility of the assays. To date, a breast cancer molecular classification panel and a colorectal cancer metastatic panel were developed and are currently being optimised for the clinical workflow by Omnigene Medical Technologies Ltd through the miniaturisation and automation of the RNA-bead plex assay.
The Innoplex RNA-bead plex assays utilise the Quantigene branched-DNA technology that runs on the Luminex xMAP technology. Specific probes are conjugated to paramagnetic microspheres (beads) that are internally infused with specific portions of red and infrared fluorophores, used by the Luminex optics (first laser/detector) to identify the specific beads known to harbour specific probes. The Quantigene branched-DNA technology builds a molecular scaffold on the specifically bound probe-target complex to amplify the signal that is read by a second laser/LED.
The workflow of the assay can be divided into a pre-analytical phase involving the lysis/homogenisation of the tissue or cells, and the analytical phase that involves hybridisation, pre-amplification and signal amplification with a total hands-on time of two hours. This is comparable to the time required to prepare a 5-plex qRT-PCR reaction. Increased multiplexing within a reaction will result in an increase in hands-on time for qRT-PCR, while the same two hours are retained for the Innoplex assays. As shown by Scerri et al, qRT-PCR 40-plex reactions will require nine hours to prepare as compared to the bead-based assay, which retains a two-hour workflow. Hence, the bead-based assays have the advantage for high-throughput analysis in multiplex format.
Performance of the Innoplex assay
We have shown in previous studies, using breast cancer patient material, that gene expression can be measured using our RNA-based multiplex assays in formalin fixed paraffin embedded patient archival material that was of low quality and low input. Using a 40-plex assay, we show that receptor status and heterogeneity in formalin fixed paraffin embedded tissue can be measured using stained micro dissected material. Comparison with the reference methods, using snap-frozen and FFPE tissues derived from patient and xenograft samples, the bead-based multiplex assays outperformed the qRT-PCR when using FFPE-tissue- derived RNA, giving reliability coefficients of 99.3-100 per cent as compared to 82.4-95 per cent for qPCR results, indicating a lower assay variance.
Multiplexing provides both sensitivity and versatility in biomarker validation and was instrumental in our hands to measure RNA transcripts in cells at low abundance, mimicking the isolation of circulating tumour cells from blood. In this study, we show that measurement of EPCAM, KRT19, ERBB2 and FN1 maintain a linear signal, down to 15 cells or less. In addition, the simple workflow with direct measurement using lysed cells, enables this assay to be translated more efficiently to clinical setting.
In conclusion, the innovative multiplex assays enable precise and personalised treatment taking into account heterogeneity of primary tumour, progression of tumour during therapy and the metastatic surveillance of the individual patient. The versatility of the method allows the development of various assays to support different applications. Our first innovative methods were developed for the molecular classification of luminal and basal breast cancer and to predict sensitivity to specific therapy in triple negative breast cancer subtype. The multiplex assays have a wide range of possible applications in the diagnosis of tumours and surveillance of tumours during therapy.
The main advantages of these methods include (a) implementation of high throughput analysis, which has a positive impact on remote testing and implementation of such assays in patient surveillance and clinical trials, (b) the digitalised result excludes subjectivity and equivocal interpretation, which are common events in image-based measurements, and also eliminates the need for highly specialised facilities and human resources, (c) accurate and precise detection of multiple targets in one assay, minimising the use of precious patient samples and (d) enables the measurement of gene expression in heterogeneous tumours and low input/low-quality patient material.
The method is streamlined with the current pathology laboratory practices resulting in a workflow that is cost-effective and with minimal turnaround time.
References available on request.