Challenges in the Commercialization of Array-Based Tests

Introduction: Accurate and non-invasive monitoring of renal allografts post-transplant is essential for early detection of acute rejection (AR) and to impact the long-term survival of the transplant. We present the development and validation of a noninvasive, spot urine-based diagnostic assay, based on measurements of six urinary DNA, protein, and metabolic biomarkers. Materials and Methods: Male and female, adult and pediatric, patients receiving a kidney from related, unrelated living donors, or unrelated deceased donors were included. Markers were selected based on legacy urine genomic, metabolomic, and proteomic studies conducted by our group. For microwell-based measurement of cfDNA, we developed a 5’ biotinylated oligonucleotide complementary chemiluminescent immunoprobe for the measurement of specific target cfDNA fragments, as previously described. Streptavidin-HRP (R&D Systems) and SuperSignal ELISA Femto Substrate (Thermo Fisher Scientific) were used for luminescent detection and quantitation. CXCL10, clusterin, and methylated cfDNA (m-cfDNA) were measured using custom-developed assays. Total protein was measured using the Pierce Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scientific). Urinary creatinine was used to normalize the five other biomarkers for urinary concentration and hydration status and was measured using the Creatinine Assay Kit (BioAssay Systems). Results: The performance of this assay for detecting kidney injury in both native kidneys and renal allografts is presented on a cohort of 601 distinct urine samples. The urinary composite score enables diagnosis of AR, with a receiver-operator characteristic (ROC) curve area-under-the-curve (AUC) of 0.99. In addition, we demonstrate the clinical utility of this assay for predicting AR, prior to a rise in the serum creatinine, enabling earlier detection of rejection than currently possible by standard of care tests.Conclusion: This noninvasive, sensitive, and quantitative approach is a robust and informative method for the rapid and routine monitoring of renal allografts.

Introduction: Standard of care (SOC) in managing malignant gastrointestinal bleeding (GIB), including traditional endoscopic, radiological, surgical and radiotherapeutic approaches, is limited in hemostatic effectiveness. TC-325 is a novel endoscopic hemostatic powder that has shown promise in managing such patients. Methods: Adult patients were randomized at urgent endoscopy to TC-325 or SOC if they presented with upper or lower GIB and a known luminal GI malignancy confirmed to be the source of bleeding. The primary outcome was immediate hemostasis (absence of bleeding for at least 3 minutes after endoscopic therapy). Secondary outcomes included clinical rebleeding, transfusion use, ICU, LOS, additional treatment and mortality. Results: A pre-planned 20 patients were randomized to TC-325 or SOC (25% female, age 67.2±15.9 years, 2.9±1.7 comorbidities). Adopted research methods performed well in securing informed consent and measuring pertinent clinical outcomes. An initial source of bleeding was found in 85% of patients, with oozing noted in 95.0%. Immediate hemostasis was achieved in 90% (95%CI: 59.6;98.2) of patients treated initially with TC-325 versus 40% (16.8;68.7) in the SOC group. After failed immediate hemostasis with SOC, 83.3% (43.7; 97.0) of patients crossed over to TC-325, with hemostasis then being achieved at the index endoscopy in 80% (37.6;96.4) of these cross-overs. None of the TC-325 patients crossed over to SOC (the sole patient having failed TC-325 died of exsanguination at index gastroscopy from rupture of a malignant inferior mesenteric artery aneurysm before other endoscopic modalities could be attempted). Overall, hemostasis at index endoscopy was achieved in 87.7% (62.1;96.3) whether initial treatment with TC-325 or following SOC failure. Rebleeding was lower in the TC-325 group (22.2% (0;56.1)) compared to patients treated with SOC (60% (23.1;96.9)). Other outcomes are shown in table 1. Conclusion: This pilot trial is the first to assess TC-325 in malignant bleeding and demonstrates the feasibility of a larger randomized trial with regards to recruitment and adequate powering for hemostatic outcomes. Although this trial was not designed to seek statistically significant differences, the results suggest that TC-325 may indeed be a promising hemostatic modality in managing patients with malignant bleeding both in achieving immediate hemostasis and decreased subsequent rebleeding compared to existing standard of care.Table: Table. Primary and secondary outcomes

Clinical Outcomes Associated With Robotic Repair of the Mitral Valve

Background: Early prediction of cerebral palsy (CP) using the General Movement Assessment (GMA) during the fidgety movements (FM) period has been recommended as standard of care in high-risk infants. The aim of this study was to determine the accuracy of GMA, alone or in combination with neonatal imaging, in predicting cerebral palsy (CP). Methods: Infants with increased risk of perinatal brain injury were prospectively enrolled from 2009–2014 in this multi-center, observational study. FM were classified by two certified GMA observers blinded to the clinical history. Abnormal GMA was defined as absent or sporadic FM. CP-status was determined by clinicians unaware of GMA results. Results: Of 450 infants enrolled, 405 had scorable video and follow-up data until at least 18–24 months. CP was confirmed in 42 (10.4%) children at mean age 3 years 1 month. Sensitivity, specificity, positive and negative predictive values, and accuracy of absent/sporadic FM for CP were 76.2, 82.4, 33.3, 96.8, and 81.7%, respectively. Only three (8.1%) of 37 infants with sporadic FM developed CP. The highest accuracy (95.3%) was achieved by a combination of absent FM and abnormal neonatal imaging. Conclusion: In infants with a broad range of neonatal risk factors, accuracy of early CP prediction was lower for GMA than previously reported but increased when combined with neonatal imaging. Sporadic FM did not predict CP in this study.

The aim of this study was to demonstrate that telehomecare linking homebound patients with their home health-care nurses over the plain old telephone system (POTS) provides high-quality, clinically useful, and patient satisfactory interactions. Congestive heart failure, chronic obstructive pulmonary disease, and chronic wound-care patients receiving skilled home nursing care were randomized into control (standard home health care, HHC) and two intervention (standard care plus video conferencing/Internet access; the above plus physiological monitoring) groups. Virtual visits (VVs), consisting of two-way audio and video interactions between the central site HHC nurse and the subject at home, were compared for technical quality and clinical usefulness by the HHC nurses who performed the VVs. Subject perception of telehomecare and satisfaction with their HHC were assessed over the course of the project. There were a total of 567 virtual and 1,057 actual visits conducted for the 53 subjects completing the study. The technical quality of VVs were rated at 94.7%. They were considered to be as useful as actual visits in 90.7% of cases. Subject telehomecare perception increased after experiencing the process. All subjects were satisfied with their HHC; satisfaction increased with an increasing level of telehomecare intervention. Subjects receiving physiological monitoring and video conferencing/Internet access in addition to standard care were most satisfied with their care. VVs can be conducted over POTS. Patients can use telehomecare with moderate levels of training. These programs can provide timely and quality home health nursing care with VVs augmenting traditional home visits.

Among brain tumors, glioblastoma (GBM) is the most common and the most aggressive type, and brain metastases (BMs) occur in 20%-40% of cancer patients. Even with intensive treatment involving radiotherapy and surgery, which frequently leads to cognitive decline due to doses on healthy brain tissue, the median survival is 15 months for GBM and about 6 to 9 months for BM. Despite these treatments, GBM patients respond heterogeneously as do patients with BM. Following standard of care, some patients will respond and have an overall survival of more than 30 months and others will not respond and will die within a few months. Differentiating non-responders from responders as early as possible in order to tailor treatment in a personalized medicine fashion to optimize tumor control and preserve healthy brain tissue is the most pressing unmet therapeutic challenge. Innovative computer solutions recently emerged and could provide help to this challenge. This review will focus on 52 published research studies between 2013 and 2024 on (1) the early characterization of treatment efficacy with biomarker imaging and radiomic-based solutions, (2) predictive solutions with radiomic and artificial intelligence-based solutions, (3) interest in other biomarkers, and (4) the importance of the prediction of new treatment modalities' efficacy.

With a worldwide annual incidence of 1.8 million cases, lung cancer is the most diagnosed form of cancer in men and the third most diagnosed form of cancer in women. Histologically, 80-85% of all lung cancers can be categorized as non-small cell lung cancer (NSCLC). For patients with locally advanced NSCLC, standard of care is fractionated radiotherapy combined with chemotherapy. With the aim of improving clinical outcome of patients with locally advanced NSCLC, combined and intensified treatment approaches are increasingly being used. However, given the heterogeneity of this patient group with respect to tumor biology and subsequent treatment response, a personalized treatment approach is required to optimize therapeutic effect and minimize treatment induced toxicity. Medical imaging, in particular positron emission tomography (PET), before and during the course radiotherapy is increasingly being used to personalize radiotherapy. In this setting, PET imaging can be used to improve delineation of target volumes, employ molecularly-guided dose painting strategies, early response monitoring, prediction and monitoring of treatment-related toxicity. The concept of PET image-guided adaptive radiotherapy (IGART) is an interesting approach to personalize radiotherapy for patients with locally advanced NSCLC, which might ultimately contribute to improved clinical outcomes and reductions in frequency of treatment-related adverse events in this patient group. In this review, we provide a comprehensive overview of available clinical data supporting the use of PET imaging for IGART in patients with locally advanced NSCLC.

Patient Safety Culture and Barriers to Adverse Event Reporting: A National Survey of Nurse Executives

To evaluate the storage of samples and enzyme reference materials, and to improve the commutability for inter-laboratory surveillance of activity values of lactate dehydrogenase (LD; EC 1.1.1.27) in clinical laboratory medicine and in animal veterinary medicine, we studied the electrophoretic patterns and cold lability of LD isoenzymes from tissue sources of some common vertebrate species and also from human serum sources. Among many isoenzymes from these sources, only rat LD fractions showed similar electrophoretic patterns to those of human sera, and rat LD-1 fraction was relatively cold-stable. Total LD and isoenzyme LD-1 activities in routine laboratory samples and quality-control sera were measured using eight kinds of commercially available LD assay kit, including lactate and pyruvate substrate systems. Coefficients of variation between these assay kits were markedly reduced when the activities were calculated using the partially purified rat LD-1 fraction as an enzyme reference material, compared with the activities calculated using the factor indicated in each assay kit. In the regression analysis, the intercept and slope were calculated for the regression equations obtained from 12 pairs of these assay kits. The values obtained from a small amount of human serum and control serum samples were within the 95% confidence regions of those from larger amounts of human serum samples by using the present rat LD-1 standard for measuring LD-1 activities with lactate substrate. It was evident that a cold-stable and homogeneous LD isoenzyme as an enzyme reference material might contribute to accurate measurement of activities in heterogeneous samples for inter-laboratory quality-assurance surveys in both human and animal clinical laboratory use.

Background: In advanced NSCLC pts, current guidelines recommend broad molecular profiling for the following genes EGFR, ALK, ROS1, RET, MET, ERBB2, BRAF and KRAS. Tissue may be used for genomic testing in newly diagnosed advanced NSCLC but comprehensive plasma-based genotyping is less invasive and may provide a quicker and more complete assessment. The present study was conducted to prospectively assess performance of cfDNA compared to standard of care (SOC) tissue-based genomic testing to identify guideline recommended alterations in NSCLC pts. Methods: In this prospective, multicenter study, blood samples from treatment-naïve stage IIIB-IV NSCLC pts were tested using the Guardant360 next-generation sequencing (NGS) cfDNA panel and compared with SOC tissue testing. The primary objective was to demonstrate the non-inferiority of cfDNA vs tissue analysis for the detection of guideline-recommended biomarkers (not including KRAS) in NSCLC (NI margin = 10%). The rate of incomplete tissue genotyping was defined as the proportion of pts who were biomarker negative in tissue but incompletely genotyped for all 8 guideline-referenced biomarkers. Exploratory analyses included estimation of positive predictive value (PPV) of cfDNA against tissue genotyping and the biomarker discovery rate in tissue testing alone vs tissue plus cfDNA testing. Results: 199 metastatic NSCLC pts were enrolled between August 2016-June 2017, and 185 were included in this interim analysis. The primary objective of non-inferiority was met, with 47 actionable mutations identified by cfDNA vs. 48 by tissue (p<0.002). Tissue testing was incomplete in 117/185 (63.2%, 95%CI = 55.9%, 70.2%) of pts. The majority (70%) of biomarker negative pts had tissue genotyping for 2 or fewer of the recommended biomarkers (most commonly EGFR and ALK). The PPV of cfDNA for EGFR, ALK, and MET alterations was 96.6% (95%CI = 82.2%, 99.9%). The addition of cfDNA increased the biomarker detection rate by 35.4% (95%CI= 22.2, 50.1), including those who were negative (1), not assessed (13), or quantity not sufficient (QNS) (3) for the biomarker in tissue. Conclusions: This prospective, multi-center study demonstrates that a comprehensive and highly sensitive cfDNA NGS assay is non-inferior to SOC tissue testing in the detection of guideline-recommended actionable genomic alterations in NSCLC. Moreover, the addition of cfDNA to testing algorithms at the time of diagnosis markedly improved the biomarker detection rate in tissue negative/non-assessable pts. The PPV of the cfDNA assay utilized was high, supporting the use of cfDNA in treatment selection. These results demonstrate the clinical utility of comprehensive cfDNA genotyping to identify all guideline-recommended biomarkers in newly diagnosed advanced NSCLC pts. Citation Format: Ramon Palmero, Alvaro Taus, Santiago Viteri, Margarita Majem, Enric Carcereny, Javier Garde, Enriqueta Felip, Lourdes Gomez, Andrea Malfettone, Miguel Sampayo, Noemí López, Rebecca Nagy, Matthew Jackson, Iris Faull, Daniel Dix, Niki Karachaliou, Rafael Rosell. Use of comprehensive cell-free circuating tumor DNA (cfDNA) analysis to identify genomic biomarkers in newly diagnosed advanced non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 449.

IntroductionRare diseases (RD) are collectively common and often genetic. Families value and can benefit from precise molecular diagnoses. Prolonged diagnostic odysseys exacerbate the burden of RD on patients, families and the healthcare system. Genome sequencing (GS) is a near-comprehensive test for genetic RD, but existing care models—where consultation with a medical geneticist is a prerequisite for testing—predate GS and may limit access or delay diagnosis. Evidence is needed to guide the optimal positioning of GS in care pathways. While initiating GS prior to geneticist consultation has been trialled in acute care settings, there are no data to inform the utility of this approach in outpatient care, where most patients with RD seek genetics services. We aim to evaluate the diagnostic yield, time to diagnosis, clinical and personal utility and incremental cost-effectiveness of GS initiated at the time of referral triage (pre-geneticist evaluation) compared with standard of care. Methods and analysis 200 paediatric patients referred to one of two large genetics centres in Ontario, Canada, for suspected genetic RD will be randomised into a 1:1 ratio to the intervention (GS first) or standard of care (geneticist first) arm. An unblinded, permuted block randomisation design will be used, stratified within each recruitment site by phenotype and prior genetic testing. The primary outcome measure is time to genetic diagnosis or to cessation of active follow-up. Survival analysis will be used to analyse time-to-event data. Additional measures will include patient-reported and family-reported measures of satisfaction, understanding and perceived test utility, clinician-reported measures of perceived test utility and management impact, and healthcare system utilisation and costs. Ethics and dissemination This study was approved by Clinical Trials Ontario. Results will be disseminated, at minimum, via peer-reviewed journals, professional conferences and internal reports to funding bodies. Efforts will be made to share aggregated study results with participants and their families. Trial registration number NCT06935019.

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Hepatitis C virus (HCV) remains a significant public health burden worldwide, with an approximated 170 million people infected globally. Chronic HCV infection is associated with increased risk of liver fibrosis, cirrhosis and hepatocellular carcinoma. In developed countries, it is the leading cause of liver transplantation. The previous standard of care (SOC) for HCV was composed of pegylated interferon alpha (pegIFN-[alpha]) and ribavirin (RBV) for 48 weeks. This SOC is associated with about 50% virologic response rate and with severe side effects leading to treatment discontinuation. Since 2011, several direct-acting antiviral agents (DAAs) have been approved, aiming for interferon (IFN)-sparing regimens with shorter duration and fewer side effects. However, the mechanism of action (MOA) and the kinetics of DAAs are still not known. In this Ph.D. thesis work, we first established a multiplex assay approach to compare the relative efficacy of various DAAs and the kinetics of their antiviral activities, as a potential pre-clinical measure of their clinical utility. This approach employs flow cytometry, a Gaussia luciferase reporter system, western blot analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR), limited dilution assay [tissue culture infectious dose 50 (TCID50)], and an image profiling assay that quantifies the NS5A redistribution in response to drug treatment. Using this multiplex assay approach, we evaluated three classes of DAAs, including NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). Our results suggest that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, our data demonstrate that LDV has a more pronounced effect than DCV on viral replication, assembly, and infectivity of released virus. In contrast to DAAs, host-targeting agents (HTAs) affect host factors that are involved in the viral life cycle, conferring a higher barrier to viral resistance. Using the previously established multiplex assay approach plus a strand-specific nucleic acid visualization method, we compared the inhibition kinetics, MOA, and relative potency of cyclosporin A (CsA), an HTA that targets host factor cyclophilin A (CypA), to DAAs, including LDV, DCV, DNV and SOF. Our data show that CsA suppresses viral protein production more rapidly than other DAAs and causes lipid droplets (LDs) enlarged in size. However, LDV has the fastest effect on viral RNA and NS5A redistribution and disrupts the association of double-stranded RNA (dsRNA) with NS5A. Furthermore, inhibitors of NS5A, NS3/4A, but not of NS5B, suppressed the infectivity of released virus. NS5A inhibitors LDV and DCV rapidly suppressed the infectivity of intracellular virus,suggesting that they exert effects on HCV assembly. CypA inhibitor CsA rapidly suppressed the infectivity of extracellular virus, but significantly less efficient in inhibiting the infectivity of intracellular virus, indicating that it blocks a step of the viral life cycle after viral assembly, but before, or at the step of virus release. Taken together, my Ph.D. thesis work has led to the establishment of a multiplex assay approach and shed light on the MOA and efficacy of HCV antivirals. We used this approach to compare the kinetics and the MOA of DAAs and HTA. In addition, this approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations for therapeutic interventions.

We should engage with the Code.

The ethics of global clinical trials: In developing countries, participation in clinical trials is sometimes the only way to access medical treatment. What should be done to avoid exploitation of disadvantaged populations?

While standard doses of adjuvant fluoropyrimidine-based chemotherapies are generally safe for most patients, the risk of severe adverse drug reactions (ADRs) is increased for those with dihydropyrimidine dehydrogenase deficiency (DPYD), a genetic variation that affects drug metabolism. The objective of this study was to examine the cost effectiveness of offering DPYD pharmacogenetic-guided care, where genetic testing informs personalized dosing versus the current standard of care (SoC), which involves administering fluoropyrimidine-based therapies without prior genetic screening, for local or metastatic breast cancer patients in Qatar. We developed a two-stage decision analysis, with an analytic tree model over a 6-month period, followed by a life-table Markov model over a lifetime horizon. We compared the current SoC with the alternate strategy of DPYD genetic screening in patients living in Qatar with local or metastatic breast cancer who were eligible for adjuvant fluoropyrimidine therapy. Clinical outcomes and utilities were obtained from published studies, while healthcare costs were estimated from Hamad Medical Corporation, Qatar. The short-term outcome included the incremental cost-effectiveness ratio (ICER), defined as cost per success (survival without grade III/IV ADRs) at 6 months. The long-term outcome was the ICER, defined as cost per quality-adjusted life year (QALY) gained, with a 3% annual discount rate. The study adopted a public healthcare perspective in Qatar. Sensitivity analyses were conducted to explore the impact of key input parameters on the robustness of the model. In the short-term model, at its base case, DPYD genomic screening was dominant over SoC with a mean cost-saving of QAR84,585 (95% confidence interval [CI], 45,270-151,657). This cost saving reflects the overall economic benefits associated with the implementation of DPYD genomic screening. In the long-term model, compared to the current SoC, DPYD genetic screening would result in an ICER of QAR21,107 (95% CI -59,382-145,664) per QALY gained. Based on our model, implementing DPYD genetic screening to detect DPYD mutations in breast cancer patients before therapy initiation seems to be a cost-saving and cost-effective strategy in Qatar.