Monitoring Of Therapeutic Efficacy Research Articles

Drug hypersensitivity, in vitro tools, biomarkers, and burden with COVID-19 vaccines.

Hypersensitivity reactions to drugs are increasing worldwide. They display a large degree of variability in the immunological mechanisms involved, which impacts both disease severity and the optimal diagnostic procedure. Therefore, drug hypersensitivity diagnosis relies on both in vitro and in vivo assessments, although most of the methods are not well standardized. Moreover, several biomarkers can be used as valuable parameters for precision medicine that provide information on the endotypes, diagnosis, prognosis, and prediction of drug hypersensitivity development, as well on the identification of therapeutic targets and treatment efficacy monitoring. Furthermore, in the last 2 years, the SARS‐CoV‐2 (severe acute respiratory syndrome‐coronavirus) pandemic has had an important impact on health system, leading us to update approaches on how to manage hypersensitivity reactions to drugs used for its treatment and on COVID‐19 (Coronavirus disease) vaccines used for its prevention. This article reviews recent advances in these 3 areas regarding drug hypersensitivity: in vitro tools for drug hypersensitivity diagnosis, recently identified biomarkers that could guide clinical decision making and management of hypersensitivity reactions to drugs and vaccines used for COVID‐19.

Circulating DNA and Liquid Biopsies in the Management of Patients with Cancer.

Forty-five years ago, Cancer Research published the study by Leon and colleagues in which the authors described the observation of increased levels of cell-free DNA (cfDNA) in the serum of patients with cancer as compared with healthy individuals, with the highest serum levels in patients with metastatic cancer. Most interestingly was the correlation between serum DNA concentrations and therapy outcome: Increased serum DNA levels were associated with poor response to treatment, whereas decreases in DNA levels during treatment appeared to be a sign of better prognosis. Since the discovery of the prognostic value of blood DNA, much research has been focused on the characterization of cfDNA to understand its origins and increase the sensitivity and specificity of using cfDNA as a prognostic and predictive marker in the battle against cancer. Tumor-specific cfDNA markers that were discovered include genetic alterations, chromosomal aberrations, epigenetic modifications, and DNA fragmentation size. In recent years, due to the development of highly sensitive molecular technologies, cfDNA-based assays are now being introduced into the clinic as the so called "liquid biopsy." The advantages of a liquid biopsy over traditional biopsy and imaging have led to the implementation in the clinic for early cancer detection, improved cancer staging, early detection of relapse, real-time monitoring of therapeutic efficacy, and detection of therapeutic targets and resistance mechanisms. Despite Leon and colleagues' initial skepticism about the potential diagnostic value of serum DNA, cfDNA-based liquid biopsy has become one of the most important tools for personalized cancer treatment. See related article by Leon and colleagues, Cancer Res 1977;37:646-50.

All-in-one approaches for triple-negative breast cancer therapy: metal-phenolic nanoplatform for MR imaging-guided combinational therapy

BackgroundConventional chemotherapy has poor efficacy in triple-negative breast cancer (TNBC) which is highly heterogeneous and aggressive. Imaging-guided therapy is usually combined with diverse treatment modalities, could realize the integration of diagnosis and treatments. Therefore, the primary challenge for combinational therapy is designing proper delivery systems to accomplish multiple synergistic effects.ResultsHerein, a facile nanoplatform was manufactured to fulfill the all-in-one approaches for TNBC combinational therapy. Fe3+-based metal-phenolic networks (MPNs) with bovine serum albumin (BSA) modification served as drug delivery carriers to encapsulate bleomycin (BLM), forming BFE@BSA NPs. The self-assembly mechanism, pH-responsive drug release behavior, and other physicochemical properties of this system were characterized. The potential of BFE@BSA NPs as photothermal transduction agents and magnetic resonance imaging (MRI) contrast agents was explored. The synergistic anti-tumor effects consisting of BLM-induced chemotherapy, Fenton reactions-mediated chemodynamic therapy, and photothermal therapy-induced apoptosis were studied both in vitro and in vivo. Once internalized into tumor cells, released BLM could cause DNA damage, while Fenton reactions were initiated to produce highly toxic •OH. Upon laser irradiation, BFE@BSA NPs could convert light into heat to achieve synergistic effects. After intravenous administration, BFE@BSA NPs exhibited great therapeutic effects in 4T1 tumor xenograft model. Moreover, as T1-weighted MRI contrast agents, BFE@BSA NPs could provide diagnosis and treatment monitoring for individualized precise therapy.ConclusionsA nano-system that integrated imaging and combinational therapy (chemotherapy, chemodynamic therapy and photothermal therapy) were developed to kill the tumor and monitor therapeutic efficacy. This strategy provided an all-in-one theranostic nanoplatform for MRI-guided combinational therapy against TNBC.Graphical

Exploring the role of basophil activation test in diagnosis of Dermatophagoides farinae sensitization and evaluation of therapeutic efficacy of subcutaneous immunotherapy in children

It aims to detect basophil activation ratio (%CD63+ ) in peripheral blood of children with allergic asthma and rhinitis by using flow cytometry (FCM), so as to analyse the application values and clinical relevance of the basophil activation test (BAT) in diagnosis of Dermatophagoides farinae (Derf) sensitization and monitoring therapeutic efficacy of subcutaneous immunotherapy (SCIT). It was a prospective study. From the newly diagnosed children with asthma and rhinitis in our paediatric clinic, 39 patients diagnosed Derf sensitization and 15 patients not allergic to Derf were enrolled; another 4 healthy children were taken as control group. Using Derf extracts in concentration of 1, 10μg/mL and 100μg/mL as the stimulus, BAT results were expressed as %CD63+ in diagnosis of Derf sensitization and its correlation with skin prick tests (SPT), serum total IgE (tIgE), specific IgE (sIgE), sIgE/tIgE, specific IgG4 (sIgG4), FEV1%pred in pulmonary ventilation function, exhaled nitric oxide (FeNO), children asthma control test (C-ACT) and visual analogue scale (VAS) were observed. In sensitization group, %CD63+ , sIgG4 and clinical indicators were detected again from patients who had received SCIT to analyse their internal connections. The average levels of %CD63+ in 3 concentrations showed an increasing concentration-dependent trend overall. %CD63+ in sensitization group was significantly higher than that in the other 2 groups. The analysis of ROC for Derf sensitization showed the area under the curve (AUC) for BAT in 3 concentrations was higher than that for sIgE whose AUC is 0.893. %CD63+ was positively correlated with SPT grade, sIgE, sIgE/tIgE and VAS, and negatively correlated with C-ACT. In patients receiving SCIT, %CD63+ became lower and sIgG4 level became higher than pretreatment. There was no obvious change in sIgG4 in those who had not received SCIT. BAT is a reliable and non-invasive tool for diagnosis of Derf sensitization in children with asthma and rhinitis. CD63-based BAT has clinical value to monitor outcome of SCIT, and the change in basophil activation is inherently related to induction of sIgG4.

Longitudinal amyloid cognitive composite in preclinical Alzheimer’s disease

Previous studies have developed several cognitive composites in preclinical Alzheimer disease (AD). However, more sensitive measures to track cognitive changes and therapeutic efficacy in preclinical AD are needed considering the diverse sociocultural and linguistic backgrounds. This study developed a composite score that can sensitively detect the amyloid-β (Aβ)-related cognitive trajectory of preclinical AD using Korean data. A total of 196 cognitively normal participants who underwent amyloid positron emission tomography were followed-up with neuropsychological assessments. We developed the Longitudinal Amyloid Cognitive Composite in Preclinical AD (LACPA) using the linear mixed-effects model (LMM) and z scores. The LMM was also used to investigate the longitudinal sensitivity of the LACPA and the association between time-varying brain atrophy and the LACPA. Considering the group-time interaction effects of each subtest, the Seoul Verbal Learning Test-Elderly version immediate recall/delayed recall/recognition, the Korean Trail Making Test B Time, and the Korean Mini-Mental State Examination were selected as components of the LACPA. The LACPA exhibited a significant group-time interaction effect between the Aβ+ and Aβ- groups (t=-3.288, p=0.001). Associations between time-varying LACPA and brain atrophy were found in the bilateral medial temporal, right lateral parietal, and right lateral frontal regions, and hippocampal volume. The LACPA may contribute to reduction in time and financial burden when monitoring Aβ-related cognitive decline and therapeutic efficacy of the disease-modifying agents specifically targeting Aβ in secondary prevention trials.

Mucopolysaccharidosis Type VI, an Updated Overview of the Disease.

Mucopolysaccharidosis type VI, or Maroteaux–Lamy syndrome, is a rare, autosomal recessive genetic disease, mainly affecting the pediatric age group. The disease is due to pathogenic variants of the ARSB gene, coding for the lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). The enzyme deficit causes a pathological accumulation of the undegraded glycosaminoglycans dermatan-sulphate and chondroitin-sulphate, natural substrates of ASB activity. Intracellular and extracellular deposits progressively take to a pathological scenario, often severe, involving most organ-systems and generally starting from the osteoarticular apparatus. Neurocognitive and behavioral abilities, commonly described as maintained, have been actually investigated by few studies. The disease, first described in 1963, has a reported prevalence between 0.36 and 1.3 per 100,000 live births across the continents. With this paper, we wish to contribute an updated overview of the disease from the clinical, diagnostic, and therapeutic sides. The numerous in vitro and in vivo preclinical studies conducted in the last 10–15 years to dissect the disease pathogenesis, the efficacy of the available therapeutic treatment (enzyme replacement therapy), as well as new therapies under study are here described. This review also highlights the need to identify new disease biomarkers, potentially speeding up the diagnostic process and the monitoring of therapeutic efficacy.

Assessing CAR T-Cell Therapy Response Using Genome-Wide Sequencing of Cell-Free DNA in Patients With B-Cell Lymphomas

Methods that enable monitoring of therapeutic efficacy of autologous chimeric antigen receptor (CAR) T-cell therapy will be clinically useful. The aim of this study is to demonstrate the feasibility of blood-derived cell-free DNA (cfDNA) to predict CAR T-cell therapy response in patients with refractory B-cell lymphomas. Whole blood was collected before and throughout CAR T-cell therapy until day 154. Low-coverage (∼0.4×), genome-wide cfDNA sequencing, similar to that established for noninvasive prenatal testing, was performed. The genomic instability number (GIN) was used to quantify plasma copy number alteration level. Twelve patients were enrolled. Seven (58%) patients achieved a complete response (CR); 2 (25%), a partial response. Median progression-free survival was 99 days; median overall survival was not reached (median follow-up, 247 days). Altogether, 127 blood samples were analyzed (median, 10 samples/patient [range 8-13]). All 5 patients who remained in CR at the time of last measurement had GIN <170 (threshold). Two patients who attained CR, but later relapsed, and all but one patient who had best response other than CR had last GIN measurement of >170. In 5 of 6 patients with relapsed or progressive disease, increasing GIN was observed before the diagnosis by imaging. The abundance of CAR T-cell construct (absolute number of construct copies relative to the number of human genome equivalents) also showed a trend to correlate with outcome (day 10, P = .052). These data describe a proof-of-concept for the use of multiple liquid biopsy technologies to monitor therapeutic response in B-cell lymphoma patients receiving CAR T-cell therapy.

Therapeutic Efficacy of Artemisinin-Based Combination Therapies in Democratic Republic of the Congo and Investigation of Molecular Markers of Antimalarial Resistance.

Routine assessment of the efficacy of artemisinin-based combination therapies (ACTs) is critical for the early detection of antimalarial resistance. We evaluated the efficacy of ACTs recommended for treatment of uncomplicated malaria in five sites in Democratic Republic of the Congo (DRC): artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Children aged 6-59 months with confirmed Plasmodium falciparum malaria were treated with one of the three ACTs and monitored. The primary endpoints were uncorrected and polymerase chain reaction (PCR)-corrected 28-day (AL and ASAQ) or 42-day (DP) cumulative efficacy. Molecular markers of resistance were investigated. Across the sites, uncorrected efficacy estimates ranged from 63% to 88% for AL, 73% to 100% for ASAQ, and 56% to 91% for DP. PCR-corrected efficacy estimates ranged from 86% to 98% for AL, 91% to 100% for ASAQ, and 84% to 100% for DP. No pfk13 mutations previously found to be associated with ACT resistance were observed. Statistically significant associations were found between certain pfmdr1 and pfcrt genotypes and treatment outcome. There is evidence of efficacy below the 90% cutoff recommended by WHO to consider a change in first-line treatment recommendations of two ACTs in one site not far from a monitoring site in Angola that has shown similar reduced efficacy for AL. Confirmation of these findings in future therapeutic efficacy monitoring in DRC is warranted.

In vivo efficacy and safety of artemether\u2013lumefantrine and amodiaquine\u2013artesunate for uncomplicated Plasmodium falciparum malaria in Mozambique, 2018

BackgroundArtemisinin-based combination therapy (ACT) has been the recommended first-line treatment for uncomplicated malaria in Mozambique since 2006, with artemether–lumefantrine (AL) and amodiaquine–artesunate (AS–AQ) as the first choice. To assess efficacy of currently used ACT, an in vivo therapeutic efficacy study was conducted.MethodsThe study was conducted in four sentinel sites: Montepuez, Moatize, Mopeia and Massinga. Patients between 6 and 59 months old with uncomplicated Plasmodium falciparum malaria (2000–200,000 parasites/µl) were enrolled between February and September of 2018, assigned to either an AL or AS–AQ treatment arm, and monitored for 28 days. A Bayesian algorithm was applied to differentiate recrudescence from new infection using genotyping data of seven neutral microsatellites. Uncorrected and PCR-corrected efficacy results at day 28 were calculated.ResultsTotals of 368 and 273 patients were enrolled in the AL and AS–AQ arms, respectively. Of these, 9.5% (35/368) and 5.1% (14/273) were lost to follow-up in the AL and AS–AQ arms, respectively. There were 48 and 3 recurrent malaria infections (late clinical and late parasitological failures) in the AL and AS–AQ arms, respectively. The day 28 uncorrected efficacy was 85.6% (95% confidence interval (CI) 81.3–89.2%) for AL and 98.8% (95% CI 96.7–99.8%) for AS–AQ, whereas day 28 PCR-corrected efficacy was 97.9% (95% CI 95.6–99.2%) for AL and 99.6% (95% CI 97.9–100%) for AS–AQ. Molecular testing confirmed that 87.4% (42/48) and 33.3% (1/3) of participants with a recurrent malaria infection in the AL and AS–AQ arms were new infections; an expected finding in a high malaria transmission area. Adverse events were documented in less than 2% of participants for both drugs.ConclusionBoth AL and AS–AQ have therapeutic efficacies well above the 90% WHO recommended threshold and remain well-tolerated in Mozambique. Routine monitoring of therapeutic efficacy should continue to ensure the treatments remain efficacious.Trial registration Clinicaltrials.gov: NCT04370977

Application of LDH assay for therapeutic efficacy evaluation of ex vivo tumor models

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.

Abstract 596: Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer

Abstract Purpose: Circulating tumor cells (CTCs) may be novel biomarkers to predict cancer prognosis and monitor therapeutic efficacy. In the current study, we investigated the use of the time-dependent enumeration and targeted DNA sequensing of single CTCs as liquid biopsy based biomarkers of non-small cell lung cancer (NSCLC) patients for diagnosis, prognosis, therapeutic choice and outcome monitoring. Experimental Design: In a prospective study, we tested the AccuCyte® system (RareCyte®, Inc. Seattle, WA) for CTC enumeration in NSCLC using FDA criteria (CK/EpCAM+/CD45- with a nucleus). Whole blood (7.5 ml) from healthy donors was spiked with human lung adenocarcinoma (A549 cell line) cells at specific numbers (N=0, 100, 200, 1000). CTC detection and single-CTC picking was performed. NSCLC subjects' (N=20) and chronic smokers without cancer on screening low-dose CT subjects' (N=10) blood were collected (amount, method, biotechnology). For all blood samples, CTC detection and single CTC picking was performed. DNA was isolated from CTCs (N=3) and white blood cells (N=2) per sample. DNA libraries were prepared for targeted oncogene characterization using the CleanPlex® OncoZoom® panel. Somatic variant analysis and comparisons were performed. Results: AccuCyte enumeration of A549 cells spiked at four levels in healthy subjects' whole blood showed (Spiked #, Detected #): (0, 0); (100, 76); (200, 208); (1000, 1223). NSCLC subjects' (N=20; age: 64.85±6.64; BMI: 28.00±8.15sex ratio: 0.42) blood had a mean CTC count of 13.4 (SD=52, median=0, range [0, 237]). Ten screening controls without cancer (64.2±5.81, 1, 28.50±6.13, 0.66) had a mean CTC count of 0.20 (0.42, 0, [0, 1]). Significant association was noted regarding a higher number of CTCs found in NSCLC compared to screening subjects without cancer according to the (p= 0.020, Wilcoxon signed rank). CTC numbers increased with NSCLC AJCC stage. Somatic variant analysis on patient-matched single CTCs and WBCs is ongoing. Conclusions: The molecular investigation of patient matched single CTCs and white blood cells will fundamentally advance understanding on the NSCCL carcinogenic signature of these blood born cells and their potential value as cancer detection and therapy management liquid biopsy-based biomarkers. Citation Format: Mouadh Barbirou, Yariswamy Manjunath, Amanda Miller, Arturo Ramirez, Nolan Ericson, Kevin F. Kevin F. Staveley-O'Carroll, Wes Warren, Guangfu Li, Peter J. Tonellato, Jussuf T. Kaifi. Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 596.

Abstract LB053: Clinical evaluation of NGS-based liquid biopsy genotyping in non-small cell lung cancer (NSCLC) patients

Abstract Background: Access to molecular testing for metastatic NSCLC (mNSCLC) patients has been improved by circulating tumor DNA (ctDNA) based liquid biopsies (LB), which can obviate invasive procedures, expedite results, and enable serial testing. Emerging clinical applications for LB include therapeutic efficacy monitoring and minimal residual disease (MRD) detection, which demand a ctDNA assay with high sensitivity, specificity, and appropriate genomic coverage. Here we demonstrate that SafeSEQ next-generation sequencing (NGS) LB delivers equivalent performance to OncoBEAM digital PCR, a sensitive ctDNA approach that has been extensively clinically validated in pivotal trials (e.g., AURA, TIGER-X). Importantly, SafeSEQ delivers significantly expanded genomic coverage to address the need for expanding targeted therapy indications, monitoring treatment response, and detecting MRD. Methods: Whole blood samples (n=176) were collected from mNSCLC patients prior to/during treatment or at disease progression and transported to a CLIA laboratory for OncoBEAM analysis to detect mutations in EGFR (exon 19 del, L858R, T790M, C797S), KRAS (codons 12, 13, 61) and BRAF V600E. Replicate plasma aliquots were analyzed with SafeSEQ to interrogate clinically relevant regions in BRAF, EGFR, ERBB2, KRAS, MET, NRAS, PIK3CA, and TP53. Mutation level concordance between the methods was assessed, where only genomic alterations interrogated by both platforms were considered. For mutations detected by both methods, correlation analysis of mutant allelic frequency (MAF) was performed. Results: Concordance analysis of the mutation results from OncoBEAM and SafeSEQ testing of 176 replicate patient samples demonstrated an overall percent agreement (OPA) of 99.6%, with a positive percent agreement (PPA) of 78.1% and a negative percent agreement (NPA) of 99.9%. The mean MAF for discordant mutations (n=16) was 0.06% (range: 0.04-0.12%). When considering mutations with MAF &amp;gt;0.1%, PPA and OPA increased to 96.0% and 99.9%, respectively. MAF levels for mutations detected by both methods demonstrated a strong linear correlation (R2=0.98). Of 124 patient samples having no mutation detected by OncoBEAM, 75 (60%) showed ≥1 alteration with SafeSEQ. Panel-wide, 76% and 30% of all mutations were detected at &amp;lt;1% and &amp;lt;0.1% MAF, respectively. Conclusions: SafeSEQ demonstrates clinical sensitivity comparable to OncoBEAM, with a strong positive correlation between MAF values across a broad dynamic range. SafeSEQ also provides expanded coverage across broader genomic regions, which - when combined with robust clinical performance - should better inform treatment selection, improve high resolution monitoring of therapeutic efficacy, and enable MRD detection and surveillance for NSCLC patients. Citation Format: Hillary Sloane, Priya Sathyanarayan, Daniel Edelstein, Frederick Jones, Jennifer Preston, Sam Wu, Jenna Los, Lara Duchstein, Johannes Fredebohm, Katharina Wichner, Denise Heim, Frank Holtrup, Hannah Quinn, David Feller-Kopman. Clinical evaluation of NGS-based liquid biopsy genotyping in non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB053.

Systemic Mastocytosis: Molecular Landscape and Implications for Treatment.

Over the past decade, we have witnessed significant advances in the molecular characterization of systemic mastocytosis (SM). This has provided important information for a better understanding of the pathogenesis of the disease but has also practically impacted the way we diagnose and manage it. Advances in molecular testing have run in parallel with advances in therapeutic targeting of constitutive active KIT, the major driver of the disease. Therefore, assessing the molecular landscape in each SM patient is essential for diagnosis, prognosis, treatment, and therapeutic efficacy monitoring. This is facilitated by the routine availability of novel technologies like digital PCR and NGS. This review aims to summarize the pathogenesis of the disease, discuss the value of molecular diagnostic testing and how it should be performed, and provide an overview of present and future therapeutic concepts based on fine molecular characterization of SM patients.

Observation of varicella zoster virus-induced acute retinal necrosis: viral load detection and visual outcome.

To observe the changes of viral load in aqueous humour samples and visual outcomes in varicella zoster virus (VZV)-induced acute retinal necrosis (ARN). Observational retrospective study. Medical records and viral load measured by real-time quantitative polymerase chain reaction (qPCR) of 20 eyes with VZV-induced ARN were reviewed. The mean viral load at presentation was 5.7 × 107 ± 9.7 × 107 copies/mL. An initial plateau phase for viral load lasting up to 2 weeks occurred in most eyes (18 eyes, 90%). In the following logarithmic reduction phase, the mean slope of the decline in viral load was -0.103 ± 0.029 log/day, and the expected time for half reduction of the initial viral load was 3.2 ± 1.0 days. At the end of the first 8-week's antiviral treatment, the viral load was below detection threshold in all 20 eyes (100.0%). The mean logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) improved from 1.1 ± 0.7 (Snellen equivalent 20/250) to 0.7 ± 0.6 (Snellen equivalent 20/100) after a follow-up of 8.6 ± 2.0 months. Thirteen of the 20 eyes (65.0%) suffered retinal detachment and underwent vitrectomy. The initial viral load was the independent predictive factor of logMAR BCVA at the last follow-up (β = 0.745, P < 0.001). The observation of viral load changes by qPCR was useful for better monitoring of therapeutic efficacy and deciding needed antiviral duration in VZV-induced ARN patients.

MO250URINARY DICKKOPF-3 (UDKK3): A NEW BIOMARKER FOR LONG-TERM CKD PROGRESSION AND MORTALITY?*

Abstract Background and Aims Kidney fibrosis has been reported to be a key progression hallmark of chronic kidney disease (CKD). It seems likely that a precise biomarker of renal fibrosis extension would contribute to predict accurately the risk of a decline in glomerular filtration rate (eGFR). Previous studies have shown that the assessment of urinary Dickkopf-3 (uDKK3), a stress induced tubular epithelial-derived profibrotic glycoprotein, might be a potential tubulointerstitial fibrosis biomarker and might identify patients at short-term risk of eGFR loss. We aim to evaluate uDKK3 as a potential biomarker for long-term CKD progression in a cohort with various etiologies of CKD, and subsequently in an overt diabetic nephropathy cohort. We also tested the role of treatment with RAAS blockers on the uDKK3 levels and if the treatment could modify them. Method We prospectively studied two independent cohorts consisted of 356 patients with stage 2-3 CKD. Progreser cohort comprised 255 patients with heterogeneous etiologies of CKD and Pronedi cohort 101 patients with overt diabetic nephropathy. The primary outcome was the time to the first event of the composite endpoint (&amp;gt;50% increase in serum creatinine concentration, end-stage kidney disease [ESKD], or death). We divided patients into tertiles according to their baseline uDKK3 levels: less than 1092 pg/mg (Tertile 1, T1), between 1092-5050 pg/mg (Tertile 2, T2) and higher than 5050 pg/mg (Tertile 3, T3). We used the cut point of T3 as an exploratory cut-off. Cox regression models were used to adjust for potential effects of confounders or modifiers: age, gender, mean arterial pressure, body mass index (BMI), urine albumin/creatinine ratio, urine protein/creatinine ratio and serum creatinine. Mixed-effects models were adjusted to study longitudinal data. Results Progreser cohort and Pronedi cohort did not differ in their clinical baseline characteristics except in proteinuria. At baseline, uDKK3 levels were not different between different etiologies or both cohorts, median uDKK3 was 2199 (IQR: 658-7618) pg/mg in the Progreser cohort and 3041 (IQR: 653-9777) pg/mg in the Pronedi cohort (p:0.56). Median time of follow-up was 36 months. Forty-nine patients (19 %) in Progreser cohort and 31 patients (31%) in Pronedi cohort reached the primary composite outcome. Baseline uDKK3 was significantly higher in patients who reached primary outcome. In Cox multivariate model, after adjustment for potential confounders, the highest levels of uDKK3 were found to be an independent factor for renal progression in Progreser cohort (HR 1.83, CI95% 1.11-3.31) and in Pronedi cohort (HR 2.74, CI95% 1.09-6.98). Both in the Progreser and the Pronedi cohorts, uDKK3 levels above 5050 pg/mg, were associated with a lower eGFR, higher proteinuria and were independently associated with a worse renal survival. uDKK3 gradually increased in the following months, especially in patients with higher proteinuria. Treatment with RAAS-blockers did not modify uDKK3 after 4 or 12 months of treatment. Conclusion In this study, uDKK3 ratio identified patients at high risk for long-term kidney disease progression regardless of the etiology of CKD. The hypothesis was generated in a cohort of patients with CKD of heterogeneous etiologies and was further confirmed in a cohort with overt diabetic nephropathy. The predictive role of uDKK3 persisted after adjusting by eGFR and proteinuria. uDKK3 is the first non-invasive biomarker of renal fibrosis and might serve as a useful biomarker for kidney disease progression. Therefore uDKK3 could be used by clinicians to optimize staging for renal progression and monitor therapeutic efficacy of different measures to halt CKD progression.

Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by a Five Circulating Tumor DNA Methylation Signature in Colorectal Cancer

Purpose: To develop a circulating tumor DNA (ctDNA) methylation signature for early detection of metastatic relapse and surveillance of therapeutic efficacy in stage III CRC. Perimental design: The deferentially methylated locations (DMLs) between 52 normal intestinal mucosa and 134 stage III CRC tissue and between plasma from 96 stage I/II CRC patients and 73 stage IV CRC patients were detected by high-throughput targeted methylation sequencing. CtDNA methylation signature was established by random forest with DMLs shared by plasma and tissue samples. This signature was then applied to predict the risk of metastatic disease recurrence in blood samples from 50 stage III CRC patients and dynamic monitor the recurrence risk in serial blood samples from 4 CRC patients. Results: 2237 and 1185 hypermethylation markers were identified in CRC tissue and plasma from stage IV CRC patients, respectively. Our signature achieved the sensitivity of 84.21% and specificity of 81.81% in distinguishing late CRC from early CRC in the validation cohort. Moreover, this signature yielded high sensitivity (87.5%) and specificity (94.12%) for predicting metastatic disease relapse in a cohort of 50 stage III CRC patients, and could be an independent risk factor for relapse in those patients (HR, 18.7; 95% CI, 5.3-66.2; P = 9.86e-6). The analyses of serial blood samples of CRC showed that this model was more sensitive to monitor treatment response than imaging examination and serum carcinoembryonic antigen (CEA). Conclusions: We identify a ctDNA methylation signature for stratifying risk of recurrence in stage III CRC and non-invasively monitoring treatment response.Statement of translational relevanceWe successfully developed a ctDNA methylation signature for risk assessment of metastatic disease recurrence in stage III CRC patients and surveillance of therapeutic efficacy of CRC. Funding: This work was supported by the National Key R&D program of China (2017YFC1309002), National Natural Science Foundation of China (81672821, 81872041, 81773101, 81802306, 81903002), Natural Science Foundation Key program of Guangdong Province of China (2018B0303110017), Natural Science Foundation of Guangdong Province of China (2018A030310457, 2019A1515011436), Science and Technology Planning Project of Guangdong Province, China (2017B020226005). Declaration of Interest: The authors Jianbing Fan, Xia Li are employees of AnchorDx Medical Co., Ltd., a company that focuses on the development of next generation sequencing diagnostic products for cancer detection using liquid biopsy. All other authors declare no competing financial interest. Ethical Approval: It was approved by the Human Research Ethical Review Board in Southern Hospital of Southern Medical University and Southern Theater General Hospital of Chinese people's Liberation Army.

Liquid Biopsy: From Discovery to Clinical Application.

Over the past 10 years, circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) have received enormous attention as new biomarkers and subjects of translational research. Although both biomarkers are already used in numerous clinical trials, their clinical utility is still under investigation with promising first results. Clinical applications include early cancer detection, improved cancer staging, early detection of relapse, real-time monitoring of therapeutic efficacy, and detection of therapeutic targets and resistance mechanisms. Here, we propose a conceptual framework of CTC and ctDNA assays and point out current challenges of CTC and ctDNA research, which might structure this dynamic field of translational cancer research. SIGNIFICANCE: The analysis of blood for CTCs or cell-free nucleic acids called "liquid biopsy" has opened new avenues for cancer diagnostics, including early detection of tumors, improved risk assessment and staging, as well as early detection of relapse and monitoring of tumor evolution in the context of cancer therapies.

Biomarkers for Chimeric Antigen Receptor T Cell Therapy in Acute Lymphoblastic Leukemia: Prospects for Personalized Management and Prognostic Prediction.

Chimeric antigen receptor (CAR) T cell therapy represents a breakthrough in immunotherapy with the potential of ushering in a new era in cancer treatment. Remarkable therapeutic response and complete remission of this innovative management have been observed in patients with relapse/refractory acute lymphoblastic leukemia. With CAR-T cell therapy becoming widely used both in multicenter clinical trials and as a commercial treatment, therapeutic efficacy monitoring and management of toxicities will be indispensable for ensuring safety and improving overall survival. Biomarkers can act not only as effective indicators reflecting patients’ baseline characteristics, CAR-T cell potency, and the immune microenvironment, but can also assess side effects during treatment. In this review, we will elaborate on a series of biomarkers associated with therapeutic response as well as treatment-related toxicities, and present their current condition and latent value with respect to the clinical utility. The combination of biomarker research and CAR-T cell therapy will contribute to establishing a safer and more powerful monitoring system and prolonging the event-free survival of patients.

Prognostic Significance of Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer

Simple Summary“Liquid biopsy”, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. Our results revealed for the first time a significantly higher positivity of all markers in EpCAM-positive CTCs compared to plasma-derived exosomes. We report that in EpCAM-positive CTCs, CK-19, PSMA, TWIST1 expression and GSTP1 methylation are significantly correlated with worse overall survival (OS), while in exosomes, CK-8 expression and GSTP1 and RASSF1A methylation status were significantly correlated with a lower OS. We also enumerated CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and found a correlation between the CTC and tumor-derived extracellular vesicles (tdEVs) enumeration values.Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. This prospective liquid biopsy (LB) study was based on a group of 62 metastatic castration resistant prostate cancer (mCRPC) patients and 10 healthy donors (HD) as controls. Identical blood draws were used to: (a) enumerate CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and (b) analyze CTCs and paired plasma-derived exosomes at the gene expression and DNA methylation level. CTCs were enumerated using CellSearch in 57/62 patients, with values ranging from 5 to 854 cells/7.5 mL PB. Our results revealed for the first time a significantly higher positivity of gene expression markers (CK-8, CK-18, TWIST1, PSMA, AR-FL, AR-V7, AR-567 and PD-L1 mRNA) in EpCAM-positive CTCs compared to plasma-derived exosomes. GSTP1, RASSF1A and SCHLAFEN were methylated both in CTC and exosomes. In CTCs, Kaplan–Meier analysis revealed that CK-19 (p = 0.009), PSMA (p = 0.001), TWIST1 (p = 0.001) expression and GSTP1 (p = 0.001) methylation were correlated with OS, while in exosomes GSTP1 (p = 0.007) and RASSF1A (p = 0.001) methylation was correlated with OS. Our direct comparison study of CTCs and exosomes at gene expression and DNA methylation level, revealed for the first time a significantly higher positivity in EpCAM-positive CTCs compared to plasma-derived exosomes. Future perspective of this study should be the evaluation of clinical utility of molecular biomarkers in CTCs and exosomes on independent multicentric cohorts with mCRPC patients.

Continued Low Efficacy of Artemether-Lumefantrine in Angola in 2019.

Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated Plasmodium falciparum infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers. Samples from treatment failures were genotyped for the pfk13, pfcrt, and pfmdr1 genes. Day 3 clearance rates were ≥95% in all arms. Uncorrected day 28 Kaplan-Meier efficacy estimates ranged from 84.2 to 90.1% for the AL arms and 84.7 to 100% for the ASAQ arms. Corrected day 28 estimates were 87.6% (95% confidence interval [CI], 81 to 95%) for the AL arm in Lunda Sul, 92.2% (95% CI, 87 to 98%) for AL in Zaire, 95.6% (95% CI, 91 to 100%) for ASAQ in Zaire, 98.4% (95% CI, 96 to 100%) for AL in Benguela, and 100% for ASAQ in Benguela and Lunda Sul. All 103 analyzed samples had wild-type pfk13 sequences. The 76T pfcrt allele was found in most (92%; 11/12) ASAQ late-failure samples but in only 16% (4/25) of AL failure samples. The N86 pfmdr1 allele was found in 97% (34/35) of treatment failures. The AL efficacy in Lunda Sul was below the 90% World Health Organization threshold, the third time in four rounds that this threshold was crossed for an AL arm in Angola. In contrast, the observed ASAQ efficacy has not been below 95% to date in Angola, including this latest round.