The European Organisation for Research and Treatment of Cancer (EORTC) Brain Tumor Group and Protagen AG today announced a collaboration to utilize Protagen’s Cancer Immunotherapy Array to identify autoantibody biomarkers that investigate the immunological profile and immuno-competence of long-term Glioblastoma survivors.
Glioblastoma is the most common glial brain tumor with an annual incidence above 3 per 100,000 population. The overall prognosis of glioblastoma patients remains poor. According to population-based data, median overall survival (OS) is still in the range of only one year and long-term survival is rare. However, a minority of glioblastoma patients survive for more than 60 months and these individuals are referred to as long-term survivors. The US-based Brain Tumor Funders Collaborative (BTFC) is supporting a large international research program that aims at better understanding which individuals with glioblastoma will ultimately become long-term survivors.
Through the present new collaboration, Protagen and the EORTC Brain Tumor Group will utilize Protagen’s Cancer Immunotherapy Array to understand the immunological profile of such patients to learn how to predict such long-term survival and potentially define novel pathways for therapeutic intervention.
Prof. Michael Weller, Head of the Brain Tumor Center at University Hospital Zurich and Chairman of the EORTC Brain Tumor Group, stated: “In our network we have followed and investigated this group of long-term glioblastoma survivors for many years. The focus has been to understand the molecular profile of these patients and thus over the years we have gained a much better understanding. However, we really need to understand the immunological profile and the immuno-competence of these patients better. Thus, investigating these patients by utilizing Protagen’s Cancer Immunotherapy Array may enable us to define their immune-profile, so that we can assess their immuno-competence. This will help us, together with the data already collected, to potentially understand why these patients survive for so long and how this can be extrapolated to other patients suffering from glioblastoma.”
Dr. Peter Schulz-Knappe, Protagen’s Chief Scientific Officer, commented: “Our unique Cancer Immunotherapy Array has already demonstrated its potential for the prediction of therapeutic response and immune-related adverse events in Immuno-Oncology. The extension into Glioblastoma with a specific view to studying long-term survivors with one of the deadliest tumors provides a great opportunity to apply the Array for the prediction of survival but also to learn more about potential novel pathways for therapeutic intervention. Thus, we believe that applying our technology will result in a better understanding of the immunological profile of these long-term survivors which will benefit all patients suffering from Glioblastoma. We feel privileged that the EORTC Brain Tumor Group shares this vision, and are excited about the collaboration.”

Researchers at the University of Helsinki have developed a new mouse model of congenital anomalies of kidney and urinary tract and disease progression.
About one in every 100 babies is born with some kind of developmental anomaly in the urogenital tract. In most cases abnormalities are mild, but sometimes life-long and even life-threatening disease develops.
Infertility is another important aspect that associates with urogenital anomalies. Therefore understanding how those features occur is instrumental in developing future treatments.
To date, diseases which scientist understand the best are those caused by mutations in the proteins involved. However, in many diseases such mutations are not found, and the disease is “idiopathic” or referred as without a known cause, and maybe triggered by e.g. environmental factors.
Classically scientists have studied such cases by injecting many copies of the gene of interest into fertilized egg of an experimental animal. However, the major problem with this technique is that scientist have almost no control over where in the genome the gene lands, and what cell types start to produce the encoded protein.
By employing an unconventional genome engineering trick that increased glial cell line-derived neurotrophic factor (GDNF) production 3-6 times, scientists revealed that ureter, which allows urine produced by kidneys to enter bladder, length is regulated by GDNF levels, and that tubes connecting testicles to reproductive organs are misplaced when there is too much GDNF, resulting in infertility in males.
GDNF is a secreted protein which signals growth and survival for many types of cells. In females, too much GDNF resulted in imperforated vagina or lack of vaginal opening, resulting in infertility.
The researchers were able to trace some of those defects back to altered stem cell behaviour in the developing urogenital block and identified some signalling pathways involved. Collectively these findings provide new information on altered stem cell behaviour in the developing kidney.
University of Helsinki https://tinyurl.com/y6oag6xr

The longest FlexLab automation system ever produced by Inpeco has been designed for a mega volume reference laboratory customer, Hermes Pardini group based in Minas Gerais, Brazil. Siemens Healthineers (Inpeco’s strategic automation partner who sold the solution) is implementing the FlexLab system for Hermes Pardini’s “Enterprise” project, which will result in the largest laboratory automation platform in the world, linking more than 100 analyzers and 7 clinical specialties.
The Hermes Pardini Group is highly innovative in diagnostics medicine, and will handle 110 million sample tubes every year to address diagnostic testing needs of patients and healthcare providers from all across Brazil. The Enterprise project is expected to be operating at its full capacity in 2019, and will automate the distribution of sample tubes to nearly 100 different analyzers with a total length of 330 meters, which will include more than 70 pre- and postanalytical modules to eliminate the need for error-prone and time-consuming manual interventions within the clinical laboratory. It is to be installed in the Vespasiano site (Belo Horizonte area) in the current laboratory testing area (3,500 square meters of floor space).
“Unlike conventional laboratory set-ups, where sample tubes must be moved manually between different analyzers, our enterprise lab will employ a ‘one-touch, one workflow’ concept to eliminate the need for manual interventions, ensure sample traceability, and reduce the turnaround time to results,” said Guilherme Collares, Chief Operations Officer of the Hermes Pardini Group.
Inpeco’s High Throughput FlexLab solution will contribute to make this concept a reality, and enable providers to receive results faster, thereby enabling better patient care. The project also includes refrigerated storage units, with capacity to hold more than 1.3 million samples, and a newly developed advanced vision system, which is able to detect sample deterioration such as hemolysis, icterus or lipemia. “We’ve been in touch with Hermes Pardini since they began 2 evaluating options to replace the automation system they were using – said Andrea Pedrazzini, President of Inpeco – and we are extremely proud and pleased that such a prestigious institution has decided to adopt the largest FlexLab system ever built to fully automate their samples process, from loading to storing and disposing.”

http://www.inpeco.com/en/

UK patients with N. gonorrhoeae (GC) now have the option of receiving the cheap and easily-administered oral antibiotic ciprofloxacin thanks to updated treatment guidelines and the availability of a novel molecular diagnostic and antimicrobial susceptibility test (AST). The new test can simultaneously diagnose gonorrhoea and provide information on whether a patient will respond to ciprofloxacin.

Global management strategies for antimicrobial resistance (AMR), such as those developed by the World Health Organisation, highlight gonorrhoea as a priority infection to manage, with more effective use of diagnostic tools listed as a key focus for future development. On 24 January 2019 the Health Secretary Matt Hancock, launched the UK government’s 20-year vision to combat antibiotic resistance at the World Economic Forum at Davos, Switzerland.

Ciprofloxacin was the first line antibiotic of choice for uncomplicated gonorrhoea in the past but was relegated to an alternative treatment due to increasing resistance.

The incidence of sexually transmitted infections (STIs) including gonorrhoea is increasing around the world, with corresponding increasing rates of resistance to commonly used antibiotics. The first two cases of multidrug-resistant (MDX) N. gonorrhoeae were reported in the UK at the start of 2019. These follow the first globally reported case of gonorrhoea with high-level resistance to azithromycin and resistance to ceftriaxone in England in 2018.

Called ResistancePlus® GC, the availability of the new test is timely, as the British Association of Sexual Health and HIV (BASHH) has launched its 2019 gonorrhoea management guidelines to include the use of ciprofloxacin, provided antimicrobial susceptibility results are available prior to treatment.

The new test could be critical in the battle against antibiotic resistance, helping physicians to prescribe an effective antibiotic using a resistance-guided approach. Prior to the new 2019 BASHH guidelines, ceftriaxone was the recommended front-line treatment for gonorrhoea together with azithromycin. Ceftriaxone is a painful intramuscular injection. It has long been considered the last readily available effective antibiotic for GC, and several extensively drug-resistant strains have now been isolated exhibiting resistance to ceftriaxone and many other available treatments.

“The new guideline now recommends giving double the previously recommended dose of ceftriaxone (now 1g stat). However, they also indicate ciprofloxacin as a first-line option when antimicrobial susceptibility is known prior to treatment.
Not only is this less painful for patients, it is preferable to doctors as it is a cheaper treatment that does not require specialised staff to administer the injection.

As the UK now has the lowest reported rate of ciprofloxacin-resistant gonorrhoea at 26%, over 70% of patients could now benefit from the new guidelines and receive oral therapy if they have a test result showing ciprofloxacin susceptibility, such as is offered by the test.
ResistancePlus® GC was invented by scientists in Australia and is now able to be accessed by clinicians in the UK. It is the first commercially available molecular diagnostic test providing ciprofloxacin susceptibility information and is well placed to support current laboratory molecular testing workflows. Currently, culture methods are the routine test to determine antimicrobial susceptibility, but results can take days to produce, and many rectal (50%) and the majority of throat (70%) samples can be difficult to culture; ResistancePlus® GC can overcome these limitations.

Treating gonorrhoea as soon as possible is very important because gonorrhoea can lead to serious long-term health problems including pelvic inflammatory disease (PID) in women, that may result in infertility, and infection in the testicles in men.

SpeeDx, the company behind the new test, received CE-IVD marking for its ResistancePlus® GC assay at the end of 2018, enabling it to be adopted in all countries where CE-mark is accepted – including the UK. It is based on a PlexPCR method that detects both N. gonorrhoeae (GC) and sequences in the gyrA gene of the bacteria associated with susceptibility to ciprofloxacin, in a single test.

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