Blood-based Assay Research Articles

Leucine-rich alpha 2 glycoprotein is a new marker for active disease of tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is a global health problem. At present, prior exposure to Mtb can be determined by blood-based interferon-gamma release assay (IGRA), but active TB is not always detectable by blood tests such as CRP and ESR. This study was undertaken to investigate whether leucine-rich alpha-2 glycoprotein (LRG), a new inflammatory biomarker, could be used to assess active disease of TB. Cynomolgus macaques pretreated with or without Bacille Calmette-Guerin (BCG) vaccination were inoculated with Mtb to induce active TB. Blood was collected over time from these animals and levels of LRG as well as CRP and ESR were quantified. In the macaques without BCG vaccination, Mtb inoculation caused extensive TB and significantly increased plasma CRP and LRG levels, but not ESR. In the macaques with BCG vaccination, whereas Mtb challenge caused pulmonary TB, only LRG levels were significantly elevated. By immunohistochemical analysis of the lung, LRG was visualized in epithelioid cells and giant cells of the granulation tissue. In humans, serum LRG levels in TB patients were significantly higher than those in healthy controls and declined one month after anti-tubercular therapy. These findings suggest that LRG is a promising biomarker when performed following IGRA for the detection of active TB.

Abstract P2-12-12: MyNauseaRisk assay - Predicting chemotherapy-induced delayed nausea using a blood-based assay

Abstract BACKGROUND: Nausea and vomiting are some of the most common and distressing side effects of chemotherapy according to patients. Chemotherapy-induced nausea and vomiting (CINV) places a substantial burden on patients and their loved ones, which greatly reduces the quality of life, nutritional habits, ability to work and socialize, and adhere to treatment regimens. Therefore, assessment of CINV is an essential component of care for patients receiving chemotherapy. Multiple factors influence the incidence of CINV with the chemotherapy regimen, both type and dosage, being the primary risk factor. It is generally assumed that the strongest patient-related factors are younger age and female sex. However, reports in the literature have demonstrated that using these factors clinicians underestimate the prevalence of CINV, especially delayed nausea. Thus, there is a need for risk assessment tools to accurately identify patients requiring anti-emetic regimens to improve quality of life of patients and their families. Our assay is based on the knowledge that chemotherapeutic agents cause bursts of reactive oxygen species (ROS) resulting in cellular damage and release of substances that activate receptors in the chemoreceptor trigger zone. Glutathione (GSH), a key antioxidant, is responsible for maintaining redox homeostasis by neutralizing ROS. Therefore, we hypothesized that a patient’s risk of CINV may reflect individual variations in the efficiency to scavenge and neutralize ROS after chemotherapy. METHODS: Patients enrolled in our institutional review board approved study (N= 220), were treated in the adjuvant or neoadjuvant setting with highly or moderately emetogenic chemotherapies for their breast cancer diagnosis. Blood samples were drawn from chemotherapy naïve patients and used in the predictive blood assay. The assay is based on the conversion of a bioactive probe, hydroxyethyl disulfide, into mercaptoethanol, which once normalized to red blood cell count, indicates glutathione recycling capacity. Incidence and severity of delayed nausea symptoms were collected from notes in medical records as well as anti-emetic prescription history. RESULTS: A recent second evaluation of patients treated with platinum-based therapies confirmed our previously published data, demonstrating a correlation between low glutathione recycling capacity and risk of delayed nausea for patients with lung and colon cancer (N=111; misclassification rate 0.153; AUC 0.72). In this study, we evaluated age-matched patients treated with anthracycline/cyclophosphamide (AC)(N=42), docetaxel/cyclophosphamide (TC)(N=39), docetaxel/carboplatin/trastuzumab (TCH)(N=27) or docetaxel/carboplatin/trastuzumab/pertuzumab (TCHP)(N=25). The incidence of moderate-to-severe nausea was highest in the AC (38.1%) and TCHP (36.0%) groups. The TCH group had the lowest incidence of patients (11%) reporting delayed nausea. SAS/STAT v.14.1 classification trees demonstrated a weak association between anthracycline-induced delayed nausea and MyNauseaRisk score (misclassification rate 0.333; AUC 0.67). Similarly, patients treated with TC demonstrated a correlation between MyNauseaRisk score and severity of nausea as previously seen for platinum therapies. The algorithm determined that age stratification of patients in the TCHP group prior to applying the MyNauseaRisk score reduced the misclassification rate (0.280) and improved the AUC (0.72). CONCLUSION: The results from this exploratory prospective study suggests that a reduced ability to recycle GSH in the blood may offer an objective prediction of delayed nausea, possibly allowing for optimal anti-emetic regimen to improve the quality-of-life for breast cancer patients. Citation Format: Nicole F Laslett, Dillon D McCourt, Kinjal Parikh, John F Kennedy, Zonera A Ali, Erik L Zeger, Aarti L Shevade, Tracey L Evans, Paul B Gilman, Margaretha Wallon. MyNauseaRisk assay - Predicting chemotherapy-induced delayed nausea using a blood-based assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-12-12.

Abstract P6-10-18: Development and validation of novel biomarkers of response to radiotherapy in breast cancer

Abstract Background: Radiotherapy (RT) plays an important role in the multimodal treatment of breast cancer (BC). Despite improvements in the accuracy of delivering radiation to specific biological target volumes, the clinical response of BC to RT is still affected by intrinsic/acquired radioresistance. These resistant cancer cells can contribute to the development of recurrent disease and poor patient outcomes. Clinical signs of RT response are often not apparent for several weeks post-treatment; patients who fail to respond will therefore initially go undetected. There are currently no clinically validated biomarkers that can predict which patients will respond to RT or assess response during treatment. Our study aims to address this major clinical need through the identification and validation of biomarkers of radiation responsiveness. Methods: The effects of different radiation doses (2 - 10Gy) at a range of time points (1 - 24h) were investigated by analysing the protein secretion profiles from 3 BC cell lines: MCF-7 (ER+), ZR-751 (ER+) and MDA-MB-231 (ER-). Conditioned media was collected from each dose/time point and proteins isolated for mass spec analysis. For comparison, radioresistant models were derived from each of the 3 cell lines and were characterized by proliferation and colony formation assays, invasion and migration assays, whole-genome transcriptomic sequencing (WGTS) analysis and western blotting. To assess intrinsic response to RT a panel of 16 BC cell lines were evaluated by colony formation assays following a 2Gy dose of radiation. WGTS of a patient cohort of 230 (138 ER+ve, 92 ER-ve) post-menopausal women with BC, treated with breast conserving surgery and adjuvant RT but no systemic adjuvant therapy, with a median follow-up of 14 years, is currently underway. Results: 9 biomarkers emerged whose secretion was significantly increased with radiation. These were evaluated by western blotting of conditioned media 24h after a 2Gy dose of RT in matched radio-sensitive and resistant cell lines; this confirmed significantly higher levels of radiation induced secretion from sensitive cells compared to resistant. Radioresistant cell lines were characterised by epithelial-to-mesenchymal-transition, enhanced invasion/migration, loss of ER and PgR and increased EGFR and PI3K signaling activity. Initial mechanistic investigations suggest that biomarker release in response to radiation occurs via microvesicles. A blood-based assay to test the level of these secreted biomarkers is currently under development. Pre-treatment levels of the 9 biomarkers were also found to be associated with prediction of intrinsic response to RT at both gene and protein level. A gene expression signature comprising the 9 candidates is strongly associated with the intrinsic response to RT across the 16 BC cell lines studied, with higher expression found in those more sensitive to RT compared with those less sensitive or resistant. Validation of the predictive power of these biomarkers in terms of recurrence-free and overall BC specific survival is currently being assessed at gene and protein level in the patient cohort. Conclusions: We have identified 9 biomarkers which are released from BC cells sensitive to radiation 24h after a 2Gy dose (in line with current clinical standards) but not from radio-resistant derivatives.A blood based assay is currently under development which has the potential to monitor response to RT in the neoadjuvant and palliative settings.Intracellular levels of the 9 biomarkers are strongly associated with intrinsic response to RT and may hold predictive potential.These biomarkers may have the potential to improve patient care by identifying patients less likely to benefit from RT, paving the way for personalization of treatment, including altered dosing schedules and the future use of emerging radio-sensitizers. Citation Format: James Meehan, Mark Gray, Carlos Martinez-Perez, Charlene Kay, J Michael Dixon, Jimi Wills, Carol Ward, Alex von Kriegsheim, Niall Quinn, Olga Oikonomidou, David Cameron, Simon P Langdon, David Argyle, Ian H Kunkler, Arran K Turnbull. Development and validation of novel biomarkers of response to radiotherapy in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-18.

Abstract P5-01-20: Blood-based detection of PTEN loss and pathway alterations for personalized treatment of breast cancer

Abstract Introduction: Loss of function of the PTEN tumour suppressor, which leads to elevated PI3K/Akt pathway activation, is one of the common events in breast cancer. Although the mechanisms of PTEN loss of function are diverse, the most frequently observed events are PTEN gene deletion or mutation, resulting in little or no PTEN protein expression measured by tissue-based immunohistochemical staining. Tissue-based immunostaining or genetic analysis could be challenging due to its invasive nature, whereas a robust assay for blood-based detection of copy number loss has been lacking. The aim of this pilot study was to explore the pattern of genetic alterations of PTEN and its downstream PI3K-AKT pathways in breast cancer using a minimally-invasive, blood-based next generation sequencing (NGS) technology. Methods: Plasma samples were prepared from a cohort of 244 breast cancer (BC) samples of all stages. Cell-free DNA (cfDNA) was extracted from plasma sample and further analyzed using the PredicinePLUS™ NGS 180-gene panel. Specifically, cfDNA was enriched for targeted regions using a hybridization-based protocol, and sequenced using Illumina sequencer. Mutational profiling was analyzed using Predicine’s proprietary DeepSea bioinformatics platform with focus on coding exonic regions of genes of interests in PTEN, PI3K and AKT pathway. Results: cfDNA-based PTEN copy number loss was detected in 7/244 BC samples, whereas PTEN mutations were found in 19 out of 244 BC samples leading to loss of PTEN. Specifically, there are frameshift variants (p.Ile8MetfsTer16 and p.Arg130GlnfsTer4), stop gain variant (p.Arg233Ter, p.Glu242Ter), start lost variant (p.Met1?), inframe deletion variants (p.Ile101del), frameshift deletion variant (p.Ile168SerfsTer11), missense variants (p.Cys136Arg, p.Ile122Asn, p.Phe206Leu, p.Pro30Leu, p.Leu318Phe). Along the PI3K-AKT pathway, we also observed activating mutations in AKT1 (E17K only) in 12/244 samples, PIK3CA (p.His1047Arg, p.Glu545Lys, p.Glu542Lys, etc) in 99/244 samples, PIK3CB variants (p.Gly1031Trp, p.Arg153Cys, p.Tyr888Ter, p.Ser890Cys) in 4/244 samples. Interestingly, PTEN loss and PI3K/AKT pathway alterations were not mutually exclusive events in breast cancer. Conclusions: This study demonstrated the capacity of blood-based detection of gene deletion and genomic variants associated with loss of PTEN function in breast cancer. The findings further revealed the mutational spectra of PTEN-PI3K-AKT pathway alterations that could lead to better understanding of the biological mechanisms of PTEN loss in breast cancer. This blood-based liquid biopsy assay may ultimately result in improvement of precision diagnostics for biomarker-driven treatment options and patient prognosis in breast cancer. Citation Format: Pan Du, Eric Jia, Zhixin Zhao. Blood-based detection of PTEN loss and pathway alterations for personalized treatment of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-20.

Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease by the Plasma and Serum Amyloid-beta 42 Assay through Highly Sensitive Peptoid Nanosheet Sensor.

Alzheimer's disease (AD) is the most common neurodegenerative disorder with a continuous pathophysiological process starting from the preclinical and mild cognitive impairment (MCI) phases to the dementia phase. Early diagnosis is prerequisite for the early intervention of AD but meanwhile challenging. Amyloid-beta 1-42 (Aβ42) plays a crucial part in AD pathology. Positron-emission tomography (PET) imaging of Aβ42 in the brain and the measurement of Aβ42 in the cerebrospinal fluid (CSF) have been adopted for the auxiliary diagnosis of AD, but their widespread clinical application has been limited due to the radiation and the high-cost of PET and the invasive lumbar puncture for collecting CSF. Noninvasive and cost-effective blood-based assay is desirable for the early diagnosis of AD. Here, a label-free assay for the quantification of blood Aβ42 was developed using the high-throughput surface plasmon resonance imaging method with the aid of an antibody-mimetic peptoid nanosheet equipping Aβ42-recognizing loops. We demonstrated that this nanosheet-based sensor system could distinguish the plasma and sera from normal individuals and patients suffering AD and amnestic MCI with high sensitivity and specificity, preceding the diagnostic performance of the Aβ42-recognizing molecule and the antibody specific to Aβ42. This work provides a label-free, cost-effective, highly sensitive, and high-throughput blood-based assay for early detection of AD.

Pharmacogenomic blood-based assay to predict chemotherapy response and survival in pancreatic cancer.

738 Background: Pancreatic adenocarcinoma (PDAC) is for most patients a refractory disease. Modern cytotoxic chemotherapeutics (C) are not yet optimal for inducing responses and extending life. We are developing a blood-based pharmacogenomic (PGx) assay profiling circulating tumor and invasive cells (CTICs) to predict sensitivity and resistance to C. Methods: The PGx assay was studied in two cohorts of patients (pts) presenting for frontline C therapy of metastatic PDAC. Cohort 1 is from an ongoing prospective study (planned n=80); pts are enrolled prior to receiving either FOLFIRINOX or gemcitabine (Gem)/nab-paclitaxel (Nab-P). Cohort 2 (n=50) consists of pts enrolled prior to receiving bespoke combinations of C agents informed by the assay. 6 mL of peripheral blood was collected from pts at baseline and while on C therapy. CTICs were isolated by previously described collagen invasion assay, total RNA was extracted and gene-expression analysis was performed. PGx models for seven C agents used in PDAC were applied, and correlated to treatment received. Pts were classified as sensitive if C received were predicted to be effective and resistant if not. Objective endpoints were PFS and OS. Results: Cohort 1 patients who received sensitive first-line C combinations experienced significantly longer time to progression (TTP) v resistant (Table). In Cohort 2, the PGx assay was predictive of TTP and OS when used across multiple lines of therapy, with a two-year survival of 38%. Greater OS was observed in Cohort 2 pts receiving heterogenous C regimens more highly correlated to those predicted by the PGx assay. Conclusions: The PGx assay has promising predictive performance in both standard and personalized C regimens. A prospective, directed trial comparing these approaches is warranted. Clinical trial information: NCT03033927. [Table: see text]

Erratum: A Validation Study of a Multiple Reaction Monitoring-Based Proteomic Assay to Diagnose Breast Cancer.

[This corrects the article on p. 579 in vol. 22, PMID: 31897331.].

Application of mathematical models and microfluidics in the analysis of saliva mixing with antiseptic solutions

Background/Aim: Human saliva offers many advantages over blood-based biochemical assays, therefore, becomes the biological fluid of interest. Once antiseptic solutions react with saliva, both fluids undergo significant changes of their biophysical properties, consequently, those changes have an impact on their principal function. Material and Methods: In this study, saliva was collected and mixed with 0,1% chlorhexidine digluconate solution, fluoride mouthwash, zinc-hydroxyapatite solution and CPP-ACP paste. Microfluidic PVC/Green tape chips within the experimental setup were used to simulate solution mixing. The chip had 2 inlets and 1 outlet, and channel was designed in Y shape without any obstacles. The inlet channels were set at a 60° angle. The channel width was 600 µm and the diameter of inlets and outlet was 2 mm. For better visualization, blue food coloring was added to the saliva. The procedure was recorded with digital USB microscope camera and afterwards the percentage of mixing was obtained by MATLAB programming language. Results: Obtained results show incomplete mixing of all the solutions with saliva. The value of mixed liquid, when mixing 0,1% chlorhexidine digluconate solution with saliva was 51,11%. In case of medium concentration fluoride mouthwash, result was 84,37%. Zinc hydroxyapatite solution obtained result of 85,24%, and the fourth tested solution, CPP-ACP paste, 83,89%. Conclusions: Analyzed mouthwashes exhibit specific, non uniform behavior during mixing with saliva. Microfluidic setups could be efficiently used in simulating real clinical conditions in laboratory settings. Image processing mathematical models are applicable, accurate and useful in determination of the interaction of saliva with commonly used antiseptic solutions.

Predicting Amyloid Burden to Accelerate Recruitment of Secondary Prevention Clinical Trials

BackgroundScreening to identify individuals with elevated brain amyloid (Aβ+) for clinical trials in Preclinical Alzheimer’s Disease (PAD), such as the Anti-Amyloid Treatment in Asymptomatic Alzheimer’s disease (A4) trial, is slow and costly. The Trial-Ready Cohort in Preclinical/Prodromal Alzheimer’s Disease (TRC-PAD) aims to accelerate and reduce costs of AD trial recruitment by maintaining a web-based registry of potential trial participants, and using predictive algorithms to assess their likelihood of suitability for PAD trials.ObjectivesHere we describe how algorithms used to predict amyloid burden within TRC-PAD project were derived using screening data from the A4 trial.DesignWe apply machine learning techniques to predict amyloid positivity. Demographic variables, APOE genotype, and measures of cognition and function are considered as predictors. Model data were derived from the A4 trial.SettingTRC-PAD data are collected from web-based and in-person assessments and are used to predict the risk of elevated amyloid and assess eligibility for AD trials.ParticipantsPre-randomization, cross-sectional data from the ongoing A4 trial are used to develop statistical models.MeasurementsModels use a range of cognitive tests and subjective memory assessments, along with demographic variables. Amyloid positivity in A4 was confirmed using positron emission tomography (PET).ResultsThe A4 trial screened N=4,486 participants, of which N=1323 (29%) were classified as Aβ+ (SUVR ≥ 1.15). The Area under the Receiver Operating Characteristic curves for these models ranged from 0.60 (95% CI 0.56 to 0.64) for a web-based battery without APOE to 0.74 (95% CI 0.70 to 0.78) for an in-person battery. The number needed to screen to identify an Aβ+ individual is reduced from 3.39 in A4 to 2.62 in the remote setting without APOE, and 1.61 in the remote setting with APOE.ConclusionsPredictive algorithms in a web-based registry can improve the efficiency of screening in future secondary prevention trials. APOE status contributes most to predictive accuracy with cross-sectional data. Blood-based assays of amyloid will likely improve the prediction of amyloid PET positivity.

The Gasdermin E Gene Has Potential as a Pan-Cancer Biomarker, While Discriminating between Different Tumor Types.

Due to the elevated rates of incidence and mortality of cancer, early and accurate detection is crucial for achieving optimal treatment. Molecular biomarkers remain important screening and detection tools, especially in light of novel blood-based assays. DNA methylation in cancer has been linked to tumorigenesis, but its value as a biomarker has not been fully explored. In this study, we have investigated the methylation patterns of the Gasdermin E gene across 14 different tumor types using The Cancer Genome Atlas (TCGA) methylation data (N = 6502). We were able to identify six CpG sites that could effectively distinguish tumors from normal samples in a pan-cancer setting (AUC = 0.86). This combination of pan-cancer biomarkers was validated in six independent datasets (AUC = 0.84–0.97). Moreover, we tested 74,613 different combinations of six CpG probes, where we identified tumor-specific signatures that could differentiate one tumor type versus all the others (AUC = 0.79–0.98). In all, methylation patterns exhibited great variation between cancer and normal tissues, but were also tumor specific. Our analyses highlight that a Gasdermin E methylation biomarker assay, not only has the potential for being a methylation-specific pan-cancer detection marker, but it also possesses the capacity to discriminate between different types of tumors.

Development of a Pathway-Directed Drug Screen Platform for Cutaneous T Cell Lymphoma Using Patient-Derived Xenograft Models

Cutaneous T cell lymphoma (CTCL) is an orphan disease and represents 3% of non-Hodgkin lymphomas. Mycosis fungoides and Sézary syndrome (MF/SS) are the most common subtypes of CTCL. The development of targeted therapies for orphan diseases is challenging, but particularly so in the case of MF/SS due to the lack of reliable preclinical models for this malignancy, which is limited to the skin at the early stage, but disseminated to lymph nodes and other organs as disease progresses. Here we report a novel patient-derived xenograft (PDX) mouse model of MF/SS that recapitulates the multi-compartmental nature of CTCL and a blood-based genetic biomarker assay for quantitative monitoring of systemic tumor burden in PDX mice. The PDX models were extensively characterized and exhibit cardinal clinical and histologic features of CTCL, including erythematous scaly skin lesions and eventual lymphomatous dissemination to the spleen and other organs, and maintain the molecular characteristics of their clinical counterparts. The malignant T cells harvested from spleen of PDX mice shared identical TCR sequences and immunophenotypes with corresponding MF/SS patient donor, which featured as losing CD7 and CD26 expression in CD4+ T cells. We also developed a quantitative assay of tumor burden in PDX mice by determining the amount of human β-actin cell-free DNA (cfDNA) in the plasma. The cfDNA levels in the plasma were increased in a linear fashion and correlated with the tumor growth post-implantation. To explore the utility of this PDX model for drug testing, we searched for novel agents for CTCL by performing a high-throughput screen of selected available targeted agents and identified phosphatidylinositol 3-kinase (PI3K) as a high value target. A PI3K inhibitor was advanced to our PDX model with favorable results including disease attenuation and survival prolongation. Further experiments using isoform-specific siRNA knockdowns and isoform-selective PI3K inhibitors identified PI3K-δ as the tumor-driving isoform. Additional studies showed synergistic combination of PI3K-δ inhibitors with histone deacetylase (HDAC) inhibitors. The particularly potent combination of copanlisib and panobinostat is proposed for further clinical development. Disclosures McCormick: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Aduro BioTech,Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other; BridgeBio Pharma,Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Stock;Ownership; Caris Life Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo Co., Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; NeuroTrials,LLC: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences,Inc.: Research Funding; Leidos Biomedical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Navire: Membership on an entity's Board of Directors or advisory committees, Other: Stock;Ownership; Riptide Bioscience: Membership on an entity's Board of Directors or advisory committees; Quadriga Biosciences: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PMV Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer,Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ai:Nurix Inc: Research Funding; ADC Therapeutics: Honoraria; Bayer: Honoraria; BMS: Honoraria; Kirin: Honoraria; Immune Design: Honoraria; Seattle Genetics: Honoraria.

The plasma metabolome as a predictor of biological aging in humans

Chronological age is an important predictor of morbidity and mortality; however, it is unable to account for heterogeneity in the decline of physiological function and health with advancing age. Several attempts have been made to instead define a "biological age" using multiple physiological parameters in order to account for variation in the trajectory of human aging; however, these methods require technical expertise and are likely too time-intensive and costly to be implemented into clinical practice. Accordingly, we sought to develop a metabolomic signature of biological aging that could predict changes in physiological function with the convenience of a blood sample. A weighted model of biological age was generated based on multiple clinical and physiological measures in a cohort of healthy adults and was then applied to a group of healthy older adults who were tracked longitudinally over a 5-10-year timeframe. Plasma metabolomic signatures were identified that were associated with biological age, including some that could predict whether individuals would age at a faster or slower rate. Metabolites most associated with the rate of biological aging included amino acid, fatty acid, acylcarnitine, sphingolipid, and nucleotide metabolites. These results not only have clinical implications by providing a simple blood-based assay of biological aging, but also provide insight into the molecular mechanisms underlying human healthspan.

Development of a blood-based cancer screening assay with a novel multivariate cancer risk score (MCRS) model by integrating shallow WGS data and plasma protein markers.

45 Background: Despite the increase of our knowledge about carcinogenesis and recent successful development of innovative cancer drugs, cancer mortality has only slightly decreased in the past decades. Cancer early detection is probably the most cost-effective means to reduce cancer mortality as prognosis is much better when cancer is detected and treated at the early stage. Recently studies have demonstrated that blood-based mutation detection approaches may be effective to identify asymptomatic cancer patients from general population. Methods: Here we reported a novel multivariate cancer risk score (MCRS) model that interrogates shallow whole genome sequencing (WGS) data from cell-free DNA (cfDNA) and protein markers results in a single blood draw. Results: In a prospective clinical study consisting of 76 stage I-IV cancer patients and 152 normal individuals, the MCRS model demonstrated 60% sensitivity at 98.5% specificity. In order to validate clinical utility of the MCRS model in detection of cancer patients, we collated the data from a previous study of occult maternal malignancies from 1.93 million pregnant women undergoing NIPT test between 2016 and 2017. 466 out of the 639 pregnant women who tested positive for multiple chromosomal aneuploidies (MCAs) in the initial NIPTs (i.e. 0.06 - 0.1x WGS), have also underwent the protein markers test. Out of the 466 subjects, 39 maternal malignant cancer cases were diagnosed with a median follow-up of 399 days (IR, 303 - 487 days) by imaging and histology. The cancer patients presented a wide spectrum of cancer types, the most frequent being breast cancer (10 cases), liver cancer (8) and lymphoma (8), at stage II to IV. This subgroup of 466 subjects was selected as an independent validation cohort for our study. Through our new method, we analyzed shallow WGS and proteins data. The MCRS model allowed 28 of the 39 (71.8%) cancer cases to be identified with a positive predictive value of 73.7% and specificity of 97.7%. Conclusions: Taken together, these data demonstrate the MCRS model holds promise for detecting cancer in asymptomatic individuals, particularly in the populations at high risk of cancer.

Single-Molecule Arrays for Ultrasensitive Detection of Blood-Based Biomarkers for Immunotherapy.

Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.

Clinical Utility of a Blood-based Protein Assay on Diagnostic Colonoscopy Referrals for Elevated-risk Colorectal Cancer Patients in Primary Care.

Colonoscopies are effective in finding early stage colorectal cancer (CRC), which when found in a timely manner, dramatically improve survival rates. A significant number of at-risk patients are still not screened. We investigated the utility of a blood-based protein assay to assess for CRC in patients with elevated risk on the quality of preventive care delivered by board-certified primary care physicians (PCPs) in the United States. We report on the results of a 3-part, longitudinal, randomized controlled trial. Part 1 assessed physicians' ability to identify simulated patients at risk for CRC and found PCPs missed colonoscopy referrals for high-risk patients ~40% of the time. Part 2 randomized PCPs into control and intervention arms and demonstrated that a novel blood-based protein assay increased referral rates for a diagnostic colonoscopy when caring for simulated patients. Part 3, reported herein, compares real-world colonoscopy rates of actual patients cared for by control versus intervention physicians. Part 3 was executed to confirm whether the use of the assay demonstrated the same utility in their real world, high-risk patients as found in part 2 using simulated patients. In the simulations, physicians with access to the assay were significantly more likely to order diagnostic colonoscopies. Similarly, in real-world practice, patients were also more likely to be referred for a diagnostic colonoscopy (odds ratio, 4.57; 95% confidence interval, 1.19-17.57). An increase in CRC risk, as indicated by the assay in simulated and real-life patients, was associated with a higher likelihood of appropriate patients being referred to diagnostic colonoscopy.

Cost-Effectiveness of Screening for Nasopharyngeal Carcinoma with Plasma Epstein Barr-Virus Plasma DNA or Serology in High-Incidence Populations Globally

The incidence of nasopharyngeal carcinoma (NPC) exhibits marked geographic variation globally and is intimately associated with endemic Epstein-Barr Virus (EBV) infection. Although most patients present with locoregionally-advanced disease, several population-level screening trials using blood-based EBV assays have successfully detected most cases in the asymptomatic period. Given the rarity of NPC, it is uncertain whether screening could be cost-effective, and what the optimal screening program might be. We therefore sought to define the cost-effectiveness of three blood-based screening strategies in high-incidence regions globally, and hypothesized that screening of men and women in both high-incidence and middle-income countries could be cost-effective. Using incidence data from 340 cancer registries in 69 countries, we selected 42 high-incidence populations in 15 countries with a lifetime NPC incidence exceeding 0.25%. We developed a decision-analytic model comparing three previously-tested annual screening strategies (EBV DNA PCR + endoscopy + MRI, PCR + endoscopy, and ELISA serology + endoscopy) to no screening. Transition probabilities and stage distributions were based upon screening trials. Clinical outcomes were extracted from trial data and the AJCC 8 update, and microcosting was performed using WHO-CHOICE methods. One-way and probabilistic sensitivity analyses were performed to study the impact of age, gender, utilities, and costs. A willingness-to-pay (WTP) threshold was set at twice the local per-capita GDP. Among the 42 selected populations, screening with PCR + endoscopy + MRI, PCR + endoscopy, and ELISA + endoscopy decreased NPC-specific mortality by 62%, 53%, and 72%, respectively. These strategies thereby improved 5-year NPC-specific survival from 74% in an unscreened population to 91%, 89%, and 94%. In the base case scenario, screening with PCR + endoscopy + MRI was cost-effective at the WTP threshold in 40% of selected populations, compared to 38% with PCR + endoscopy and 67% with ELISA + endoscopy. The addition of MRI to PCR was marginally more cost-effective than endoscopy alone, but was associated with small improvements in 5-year NPC-specific survival (2%). Among populations with a lifetime incidence exceeding 0.60%, screening was always cost-effective. Moreover, screening in many middle-income and most high-income countries was also cost-effective. Sensitivity analysis informed the optimal age at first screening in each region, as well as the screening of women. Probabilistic sensitivity analysis was in close agreement with base case analyses. We observed significant reductions in NPC-specific mortality with the use of blood-based screening in high-incidence populations globally. Despite economic differences and the rarity of NPC, these strategies were cost-effective in most high-incidence regions, and could be extended to both adult men and women, generally beginning at age 40.

Brigatinib in ALK-positive NSCLC Identified Via Blood-based Assays

Brigatinib in ALK-positive NSCLC Identified Via Blood-based Assays

The Role of 5-Hydroxyindoleacetic Acid in Neuroendocrine Tumors: The Journey So Far

5-Hydroxyindole acetic acid (5-HIAA) is a surrogate marker for serotonin measurement and one of the first biochemical markers used in neuroendocrine tumors. In this review, we give a brief history of 5-HIAA and its precursor serotonin. We discuss its clinical utility and diagnostic performance in small intestinal neuroendocrine tumor and describe the challenges encountered during its analysis, historically performed in urine. The introduction of blood-based assays will help overcome some of the issues associated with its measurement in urine. The diagnostic performance of serum and plasma 5-HIAA has been shown to be comparable to that of urine 5-HIAA. Thus, analysis in either serum or plasma will provide a practical and convenient alternative to urine.

Screening with a high-precision blood-based assay for Alzheimer disease

The use of biomarkers for defining the pathobiology of Alzheimer disease (AD) has had a transformative effect on the field, facilitating disease detection prior to autopsy, as well as substantially altering clinical trial design.1 In the last 5–10 years, there has been a substantial shift toward using amyloid PET in human research, as well as clinically, to rule out AD (a negative scan meaning little or no plaque is present). Likewise, obtaining a sample of CSF via lumbar puncture provides a plethora of information on amyloid plaque burden, tau pathology, neurodegeneration, and gliosis. While amyloid PET and CSF β-amyloid (Aβ)42/Aβ40 have excellent diagnostic accuracy, and correspond well with plaque burden measured postmortem,2 it is widely recognized that access to a simple blood test for AD would change the field yet again.

Blood and guts, but in a good way

Medicine Inflammatory bowel diseases (IBDs) are chronic, sometimes debilitating, disorders of the immune system. The two major forms, Crohn's disease and ulcerative colitis, can be distinguished by endoscopy and imaging, but these methods are invasive and costly, particularly for longitudinal monitoring of disease progression and therapeutic response. Rubin et al. developed a noninvasive, blood-based assay for classification of human IBDs that monitors the subtypes and functions of circulating leukocytes that traffic to the gut in response to breakdown of the intestinal barrier. In a pilot study of 68 individuals, the authors detected immune signatures that differentiated between the two forms of IBD, identified the disease state (flare versus remission), and provided other information about interpatient variability in disease behavior. Nat. Commun. 10 , 2686 (2019).