by Prof. Godfrey Grech, Dr Stefan Jellbauer and Dr Hilary Graham
Understanding the molecular characteristics of tumour heterogeneity and the dynamics of progression of disease requires the simultaneous measurement of multiple biomarkers. Of interest, in colorectal cancer, clinical decisions are taken on the basis of staging and grade of the tumour, resulting in highly variable clinical outcomes. Molecular classification using sensitive and precise multiplex assays is required. In this article we shall explain the use of innovative methodologies using signal amplification and bead-based technologies as a solution to this unmet clinical need.
Introduction
Cancer is the leading cause of death globally, accounting for 9.6-million deaths in 2018, with 70% of cancer-related mortality occurring in low- and middle-income countries. In 2017, only 26% of low-income countries provided evidence of full diagnostic services in the public sector, contributing to late-stage presentation [1]. There are various aspects that negatively affect the survival rate of patients, including but not limited to:
(a) highly variable clinical outcome mainly due to lack of molecular classification;
(b) treatment of advanced stage of the disease mainly due to lack of, or reluctance to, screening programmes, resulting in treatment of symptomatic disease that is already in advanced stage;
(c) heterogeneity of the tumours that are undetected using representative biopsies of the tumour at primary diagnostics; and
(d) lack of surveillance of patients to detect early progression of disease and metastasis, mainly due to clinically inaccessible tumour tissue and the need of sensitive technologies to measure early metastatic events.
Colorectal cancer (CRC) represents the second most common cause of cancer-related deaths, with tumour metastasis accounting for the majority of cases. To date, treatment decisions in CRC are based on cancer stage and tumour location, resulting in highly variable clinical outcomes. Only recently, a system of consensus molecular subtype (CMS) was proposed based on gene expression profiling of primary CRC samples [2]. Organoid cultures derived from CRC samples were used in various studies to adapt the CMS signature (CMS1–CMS4) to preclinical models, to study heterogeneity and measure response to therapies. Of interest, the epidermal growth factor receptor (EGFR) and receptor tyrosine-protein kinase erbB-2 (HER2) inhibitors were selective and have a strong inhibitory activity on CMS2, indicating that subtyping provides information on potential first-line treatment [3]. In CRC, copy number variations are associated with the adenoma-to-carcinoma progression, metastatic potential and therapy resistance [4]. Our recent studies using primary and matched metastatic tissue showed that TOP2A (encoding DNA topoisomerase II alpha) and CDX2 (encoding caudal type homeobox 2) gene amplifications are associated with disease progression and metastasis to specific secondary sites. Hence, introducing robust and clinically-friendly molecular assays to enable measurement of multiple biomarkers to assess matched resected material and tumour-derived cells or cell vesicles in blood during therapy and beyond, has become a necessity to overcome this deadly toll. In addition, to support diagnostics in remote countries, the assays should allow measurement in low input, low quality tissue material.
To enable precise future diagnosis and patient classification and surveillance, we developed innovative methodologies (Innoplex assays) measuring expression of multiple marker panels representing the primary tumour heterogeneity and the dynamic changes associated with disease progression. We optimized these Molecular Diagnostics Sensitive and precise multiplex assays enable accurate classification and surveillance of tumours April/May 2020 21 | methodologies for multiplex digitalized readout using various sample sources ranging from archival formalin-fixed paraffinembedded (FFPE) tissues and characterization of gene amplifications in blood-derived exosomes. In this article we summarize 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 commercialization of the assays through Omnigene Medical Technologies Ltd.
Molecular profiling technology and workflow
The Innoplex multiplex assays are based on two components, namely (a) the integration of the Invitrogen QuantiGene™ Plex Assay (Thermo Fisher Scientific) and the xMAP Luminex technology enabling multiplexing of the technique, and (b) the 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 provides the versatility of the assays. To date a breast cancer molecular classification panel and a CRC metastatic panel were developed and are currently being optimized for the clinical workflow by Omnigene Medical Technologies Ltd through the miniaturization and automation of the RNA-bead plex assay.
The Innoplex RNA-bead plex assays use 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 [5].
The workflow of the assay can be divided into a pre-analytical phase involving the lysis/homogenization of the tissue or cells, and the analytical phase that involves hybridization, pre-amplification and signal amplification with a total hands-on time of 2|h. This is comparable to the time required to prepare a 5-plex quantitative real-time (qRT)-PCR reaction. Increased multiplexing within a reaction will result in an increase in hands-on time for qRT-PCR, while the same 2|h are retained for the Innoplex assays. As shown by Scerri et al. [5], qRT-PCR 40-plex reactions will require 9|h to prepare as compared to the bead-based assay which retains a 2|h workflow. Hence, the bead-based assays have the advantage for high-throughput analysis in multiplex format.
Performance and applications
We have shown in previous studies, using breast cancer patient material, that gene expression can be measured using our RNA-based multiplex assays in FFPE patient archival material that was of low quality and low input [6]. Using a 22-plex assay, inter-run regression analysis using RNA extracted from cell lines performed well with an r2>0.99 in our hands. These assays were also evaluated by other groups using snap-frozen and FFPE tissues derived from patient and xenograft samples. In comparison with the reference methods, the bead-based multiplex assays outperformed the qRT-PCR when using FFPE-tissue-derived RNA, giving reliability coefficients of 99.3–100% as compared to 82.4–95% for qPCR results, indicating a lower assay variance [5].
One main advantage of the Innoplex assays is the direct measurement of gene expression on lysed/homogenized tissues and cells, providing a simplified workflow without RNA extraction, cDNA synthesis and target amplification. In addition, due to its chemistry and use of beads, gene expression can be measured in a multiplex format (up to 80 genes) using low input and low quality material. This enables the use of the assay in remote laboratories, and as detailed below for stained microdissected material and to measure multiple markers in low abundance material, such as blood-derived circulating tumours cells.
Comparison of gene expression data from homogenized and lysed patient tissue derived from either unstained or hematoxylin and eosin (H&E)-stained sections shows a high correlation (r2>0.98). This provides an advantage when studying heterogeneous tumours that are microdissected from H&E stained slides. In fact, using this methodology, an estrogen-receptor-positive tumour was analysed and one of the tumour foci had a more advanced tumour expressing the mesenchymal marker, FN1 (fibronectin). This was only possible by running a 40-plex assay on minimal input material (microdissected from 20|μm section) representing markers for molecular classification, epithelial to mesenchymal transition, and proliferation markers [7]. A recent audit on breast cancer diagnosis, indicates clearly that heterogeneous cases characterized using the bead-based multiplex assays on resection tumour samples are not represented in matched biopsies used for patient diagnosis. In fact, only 3.5% of 97 intra-tumour heterogeneous cases were detected in a cohort of 570 patients at diagnosis. The advantage of the digitalized result of the Innoplex assays is to avoid increasing the workload of pathologists when resected samples are re-analysed to characterize multiple sites within a tumour.
Multiplexing provides both sensitivity and versatility in biomarker validation and was instrumental in our hands to measure gene amplifications in cancer-derived exosomes (tumour-derived vesicles in blood) using plasma from CRC patients. Of interest, these methods have been optimized using cancer cell lines to measure RNA transcripts in cells at low abundance, mimicking the isolation of circulating tumour cells from blood [5]. In this study we show that measurement of transcripts of EPCAM (encoding epithelial cell adhesion molecule), KRT19 (encoding keratin, type I cytoskeletal 19), ERBB2 (encoding HER2) 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 the clinical setting. Absolute quantification of transcripts presents alternative endpoint methods to the Invitrogen QuantiGene™ Plex Assay. Droplet digital PCR (dPCR) and Nanostring’s nCounter® technology are precise and sensitive methods. Multiplexing in dPCR is limiting and RNA studies are hindered by reverse transcription inefficiency. The nCounter® technology requires multiple target enrichment (PCR-based pre-amplification) to measure low input RNA, which introduces amplification bias and risk for false positive results.
Summary
In conclusion, the innovative multiplex assays indicate a shift from reactive medicine (treating patients based on average risks) towards predictive, precise and personalized treatment that takes 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 (Figs|1 & 2). 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 [8]. As discussed above, 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 digitalized result excludes subjectivity and equivocal interpretation, which are common events in image-based measurements, and also eliminates the need for highly specialized facilities and human resources;
(c) accurate and precise detection of multiple targets in one assay, minimizing 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.
The authors
Godfrey Grech*1,2 PhD, Stefan Jelbauer3 PhD, Hilary Graham4 PhD
1 Department of Pathology, Faculty of Medicine & Surgery, University of Malta
2 Scientific Division, Omnigene Medical Technologies Ltd, Malta
3 Thermo Fisher Scientific, Carlsbad, CA 92008, United States
4 Licensed Technologies Group, Luminex Corporation, Austin, Texas

*Corresponding author
E-mail: godfrey.grech@um.edu.mt

A research team, led by investigators from Georgetown University Medical Center and Fudan University in China, has devised a very promising non-invasive and individualized technique for detecting and treating bladder cancer.
The method uses a “liquid biopsy” — a urine specimen — instead of the invasive tumour sampling needed today, and a method developed and patented by Georgetown to culture cancer cells that can reveal the molecular underpinnings of each patient’s unique bladder cancer.
Their study sets forth a cost-friendly, simpler and painless technique that can determine the best treatment for each person’s bladder tumour, monitor the progress of that treatment, predict or detect cancer recurrence early, and identify new drugs that are sorely needed for this common cancer.
“This is the first study to show, using patient samples, that a ‘living liquid biopsy’ from urine can help determine treatment. This work also suggests that we might be able to grow and test cancer cells for treatment from other ‘living biomarkers’ found in blood and saliva. We are just at the beginning of this new diagnostic innovation,” says study co-senior author Xuefeng Liu, MD, professor of pathology and oncology and member of the Center for Cell Reprogramming at Georgetown University and Georgetown Lombardi Comprehensive Cancer Center.
The ability to use a patient’s urine to grow cells is a transformational innovation from Georgetown called “conditional reprogramming,” or CR. Patient-derived cells using CR can grow indefinitely without genetic manipulation, says Liu. Before this technique, which is less than a decade old, normal cells could not grow in lab culture, and cancer cells acquired numerous genetic mutations using previous culturing techniques.
“The analysis of the mutation ratio for both patient tissue and corresponding CRC confirmed that both single nucleotide variants and DNA insertions and deletions were retained during the culturing,” says Liu.
This means that a patient’s urine produced cancer cells that molecularly matched their cancer tissue sample. “We also identified some mutations not identified in the original tumour biopsies, suggesting that the urine cell cultures better reflect overall tumour diversity than a single biopsy,” he says. “The CRC technique may also expand our understanding of how low frequency mutations help lead to bladder cancer development and progression. Overall, CRC cultures may identify new actionable drug targets and help explain why this cancer is so often resistant to treatment.”
After determining that the urine colonies and tumour tissue samples had matching molecular characteristics and genetic alterations, the researchers tested urine-based CRC cancer cells with 64 clinical oncology drugs. They found that, overall, the urine-based cancer cells were resistant to more than half of the drugs. And they discovered that many of the urine cancer cells were highly sensitive to one of the drugs, bortezomib, which is currently being tested for a different genitourinary tumour, urothelial cancer.
Georgetown University Medical Center https://tinyurl.com/y46httzz

Sartorius, a leading international partner of life science research and the biopharmaceutical industry, has supported CanSino Biologics Inc. (“CanSinoBIO”) and Maj. Gen. Chen Wei’s team at the Institute of Bioengineering at the Academy of Military Medical Sciences (“Institute of Bioengineering”) in China in their development of the first vaccine candidate against the novel coronavirus SARS-CoV-2 to enter clinical trials. CanSinoBIO and the Institute of Bioengineering used Sartorius’ BIOSTAT® STR single-use bioreactor system for the upstream preparation of the recombinant vaccine, thus ensuring the rapid linear amplification of the adenovirus vector (Ad5-nCoV) and ultimately saving time during development.
The BIOSTAT® STR single-use bioreactor system comes with updated BioPAT® toolbox for process monitoring, as well as Flexsafe® STR integrated, single-use bioprocess bags. It has been proven to be used for vaccine manufacturing because it offers rapid scalability and flexibility to adapt to fluctuating demand. The single use bags prevent cross-contamination, and reduce the time needed for washing and sanitation typical in stainless steel bioreactors. As such, the amount of time needed to prepare a vector for a vaccine is shortened from several months to (several) weeks.
“We are pleased that we can help our clients and partners accelerate vaccine development while maintaining compliance with safety protocols, thereby allowing us to contribute to better health for more people,” said Huang Xian, Head of Marketing at Sartorius BPS China.
This is the second collaboration from Sartorius, CanSinoBIO, and the Institute of Bioengineering to accelerate vaccine development. In October 2017, Sartorius’ BIOSTAT® STR50 bioreactor system was used during CanSinoBIO’s and the Institute of Bioengineering’s joint development of a recombinant vaccine against Ebola virus disease. This was the first registered Ebola vaccine in the world.

A chemical that highlights tumour cells has been used by surgeons to help spot and safely remove brain cancer in a trial presented at the 2018 NCRI Cancer Conference. The research was carried out with patients who had suspected glioma, the most common form of brain cancer. Treatment usually involves surgery to remove as much of the cancer as possible, but it can be challenging for surgeons to identify all of the cancer cells while avoiding healthy brain tissue.

Researchers say that using the fluorescent marker helps surgeons to distinguish the most aggressive cancer cells from other brain tissue and they hope this will ultimately improve patient survival. The research was presented by Dr Kathreena Kurian, a Reader/Associate Professor in brain tumour research at the University of Bristol and consultant neuropathologist at North Bristol NHS Trust, UK. The study was led by Colin Watts, Professor of Neurosurgery and chair of the Birmingham brain cancer programme at the University of Birmingham, UK. Dr Kurian explained: “Gliomas are difficult to treat with survival times often measured in months rather than years. Many patients are treated with surgery and the aim is to safely remove as much of the cancer as possible. Once a tumour is removed, it is passed on to a pathologist who examines the cells under a microscope to see if they are ‘high-grade’, fast growing cells, or ‘low-grade’ slower growing cells. And we can plan further treatment, such as radiotherapy or chemotherapy, based on that diagnosis. “We wanted to see if using a fluorescent marker could help surgeons objectively identify high-grade tumour cells during surgery, allowing them to remove as much cancer as possible while leaving normal brain tissue intact.”

The researchers used a compound called 5-aminolevulinic acid or 5-ALA, which glows pink when a light is shone on it. Previous research shows that, when consumed, 5-ALA accumulates in fast growing cancer cells and this means it can act as a fluorescent marker of high-grade cells.

The study involved patients with suspected high-grade gliomas treated at the Royal Liverpool Hospital, Kings College Hospital in London or Addenbrooke’s Hospital in Cambridge, UK. They were aged between 23 and 77 years, with an average (median) age of 59 years. Before surgery to remove their brain tumours, each patient was given a drink containing 5-ALA.

Surgeons then used operating microscopes to help them look for fluorescent tissue while removing tumours from the patients’ brains. The tissue they removed was sent to the pathology lab where scientists could confirm the accuracy of the surgeons’ work.

A total of 99 patients received the 5-ALA marker and could be assessed for signs of fluorescence. During their operations, surgeons re-ported seeing fluorescence in 85 patients and 81 of these were subsequently confirmed by pathologists to have high-grade disease, one was found to have low-grade disease and three could not be assessed.

In the 14 patients where surgeons did not see any fluorescence, only seven tumours could be subsequently evaluated by pathology but in all these cases, low-grade disease was confirmed. Professor Watts said: “Neurosurgeons need to be able to distinguish tumour tissue from other brain tissue, especially when the tumour contains fastgrowing, high-grade cancer cells. This is the first prospective trial to show the benefits of using 5-ALA to improve the accuracy of diagnosing high-grade glioma during surgery. These results show that the marker is very good at indicating the presence and location of high-grade cancer cells.
National Cancer Research Institute www.ncri.org.uk/wp-content/uploads/2018/11/Kurian-Glioma-for-online.pdf