Quantitation of neurofilament light chain protein in serum and cerebrospinal fluid from patients with multiple sclerosis using the MSD R-PLEX NfL assay.

OBJECTIVES: Neurofilament light (NfL) chain is a marker of neuroaxonal damage in various neurological diseases. Here we quantitated NfL levels in the cerebrospinal fluid (CSF) and serum from patients with multiple sclerosis (MS) and controls, using the R-PLEX NfL assay, which employs advanced Meso Scale Discovery® (MSD) electrochemiluminescence (ECL)-based detection technology. METHODS: NfL was quantitated in samples from 116 individuals from two sites (Ottawa Hospital Research Institute and Mayo Clinic), consisting of patients with MS (n=71) and age- and sex-matched inflammatory neurological controls (n=13) and non-inflammatory controls (n=32). Correlation of NfL levels between CSF and serum was assessed in paired samples in a subset of MS patients and controls (n=61). Additionally, we assessed the correlation between NfL levels obtained with MSD's R-PLEX® and Quanterix's single molecule array (Simoa®) assays in CSF and serum (n=32). RESULTS: Using the R-PLEX, NfL was quantitated in 99% of the samples tested, and showed a broad range in the CSF (82-500,000 ng/L) and serum (8.84-2,014 ng/L). Nf-L levels in both biofluids correlated strongly (r=0.81, p<0.0001). Lastly, Nf-L measured by MSD's R-PLEX and Quanterix's Simoa assays were highly correlated for both biofluids (CSF: r=0.94, p<0.0001; serum: r=0.95, p<0.0001). CONCLUSIONS: We show that MSD's R-PLEX NfL assay can reliably quantitate levels of NfL in the CSF and serum from patients with MS and controls, where levels correlate strongly with Simoa.

Quantitation of cardiac troponin I in cancer patients treated with immune checkpoint inhibitors: a case-control study.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) cause a variety of toxicities, including immune-related adverse events (irAEs), but there are no biomarkers to predict their development. Guidelines recommend measuring circulating cardiac troponin I (cTnI) during ICI therapy to detect related cardiotoxicities. Moreover, elevated cTnI has also been associated with worse outcomes in non-cardiac patients, including cancer. Thus here, we investigated whether cTnI levels were higher in patients with irAEs. METHODS: The study consisted of three groups; 21 cancer patients undergoing ICI immunotherapies who presented with irAEs, four patients without irAEs, and 20 healthy controls. Patient samples were assessed at baseline (n=25), during ICI treatment (n=25, median=6 weeks of treatment) and at toxicity (n=6, median=13 weeks of treatment). In addition to blood high sensitivity cardiac troponin I (hs-cTnI), anti-thyroglobulin (TG) and anti-thyroid peroxidase (TPO) antibodies were also quantitated to detect thyroid dysfunction, constituting the second leading toxicity (23.8%) after pneumonitis (28.6%). RESULTS: Four patients with irAEs (n=4/21; 19%) and one without irAEs (n=1/4; 25%) showed higher hs-cTnI levels at any time-point; the remaining had physiological levels. None of these patients developed cardiotoxicity. Concurrent elevated levels of anti-thyroid antibodies and hs-cTnI were detected in one patient with thyroid dysfunction (n=1/5, 20%). However, these antibodies were also elevated in three patients (n=3/16, 19%) with non-thyroid irAEs and in up to 40% of healthy controls. CONCLUSIONS: hs-cTnI was not elevated in patients with irAEs, but larger studies are needed to confirm these observations.

Cancer Screening Companies Are Rapidly Proliferating: Are They Ready for Business?

Cancer screening has been a major research front for decades. The classical circulating biomarkers for cancer (such as PSA, CEA, CA125, AFP, etc.) are neither sensitive nor specific and are not recommended for population screening. Recently, circulating tumor DNA (ctDNA) emerged as a new pan-cancer tumor marker, with much promise for clinical applicability. ctDNA released by tumor cells can be used as a proxy of the tumor burden and molecular composition. It has been hypothesized that if ctDNA is extracted from plasma and analyzed for genetic changes, it may form the basis for a non-invasive cancer detection test. Lately, there has been a proliferation of "for-profit" companies that will soon offer cancer screening services. Here, we comment on Grail, Thrive, Guardant, Delfi, and Freenome. Previously, we identified some fundamental difficulties associated with this new technology. In addition, clinical trials are exclusively case-control studies. The sensitivities/specificities/predictive values of the new screening tests have not been well-defined or, the literature-reported values are rather poor. Despite these deficiencies some of the aforementioned companies are already testing patients. We predict that the premature use of ctDNA as a cancer screening tool may add another disappointment in the long history of this field.

Multi Cancer Early Detection by Using Circulating Tumor DNA-The Galleri Test. Reply to Klein et al. The Promise of Multicancer Early Detection. Comment on "Pons-Belda et al. Can Circulating Tumor DNA Support a Successful Screening Test for Early Cancer Detection? The Grail Paradigm. Diagnostics 2021, 11, 2171".

We recently published some concerns with new technologies which are based on circulating tumor DNA (ctDNA) for early cancer detection. Most of our published criticism, including a commentary in this journal, has focused on tests developed by the biotechnology company GRAIL (their commercial product is also known as The Galleri Test). Scientists from GRAIL provided explanations and rebuttals to our criticism. They also posed some questions. Here, we reiterate our position and provide rebuttals, explanations and answers to these questions. We believe that constructive scientific debates, like this one, can profoundly contribute to advancements in scientific fields such as early cancer detection.

Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection.

OBJECTIVES: Widespread SARS-CoV-2 testing is invaluable for identifying asymptomatic/pre-symptomatic individuals. There remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests suitable for frequent mass testing. Compared to nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling. Current saliva-based SARS-CoV-2 rapid antigen tests (RATs) are hindered by limited analytical sensitivity. Here, we report one of the first ultrasensitive, saliva-based SARS-CoV-2 antigen assays with an analytical sensitivity of <0.32 pg/mL, corresponding to four viral RNA copies/µL, which is comparable to that of PCR-based tests. METHODS: Using the novel electrochemiluminescence (ECL)-based immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 salivas, obtained from non-COVID-19 and COVID-19 patients. We then verified the results with a second, independent cohort of 689 patients (3.8% SARS-CoV-2 positivity rate). We also compared our method with a widely used point-of-care rapid test. RESULTS: In the first cohort, at 100% specificity, the sensitivity was 92%. Our assay correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were obtained for 86 cases. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (<0.32 pg/mL) and strongly positive (>2 pg/mL) N antigen concentrations. In the second cohort, at 100% specificity, sensitivity was also 92%. Our assay is about 700-fold more sensitive than the Abbott Panbio Rapid Test. CONCLUSIONS: We demonstrated the ultrasensitivity and specificity assay and its concordance with PCR. This novel assay is especially valuable when compliance to frequent swabbing may be problematic.

Harmonized D-dimer levels upon admission for prognosis of COVID-19 severity: Results from a Spanish multicenter registry (BIOCOVID-Spain study).

Coagulopathy is a key feature of COVID-19 and D-dimer has been reported as a predictor of severity. However, because D-dimer test results vary considerably among assays, resolving harmonization issues is fundamental to translate findings into clinical practice. In this retrospective multicenter study (BIOCOVID study), we aimed to analyze the value of harmonized D-dimer levels upon admission for the prediction of in-hospital mortality in COVID-19 patients. All-cause in-hospital mortality was defined as endpoint. For harmonization of D-dimer levels, we designed a model based on the transformation of method-specific regression lines to a reference regression line. The ability of D-dimer for prediction of death was explored by receiver operating characteristic curves analysis and the association with the endpoint by Cox regression analysis. Study population included 2663 patients. In-hospital mortality rate was 14.3%. Harmonized D-dimer upon admission yielded an area under the curve of 0.66, with an optimal cut-off value of 0.945 mg/L FEU. Patients with harmonized D-dimer ≥ 0.945 mg/L FEU had a higher mortality rate (22.4% vs. 9.2%; p < 0.001). D-dimer was an independent predictor of in-hospital mortality, with an adjusted hazard ratio of 1.709. This is the first study in which a harmonization approach was performed to assure comparability of D-dimer levels measured by different assays. Elevated D-dimer levels upon admission were associated with a greater risk of in-hospital mortality among COVID-19 patients, but had limited performance as prognostic test.

Can Circulating Tumor DNA Support a Successful Screening Test for Early Cancer Detection? The Grail Paradigm.

Circulating tumor DNA (ctDNA) is a new pan-cancer tumor marker with important applications for patient prognosis, monitoring progression, and assessing the success of the therapeutic response. Another important goal is an early cancer diagnosis. There is currently a debate if ctDNA can be used for early cancer detection due to the small tumor burden and low mutant allele fraction (MAF). We compare our previous calculations on the size of detectable cancers by ctDNA analysis with the latest experimental data from Grail's clinical trial. Current ctDNA-based diagnostic methods could predictably detect tumors of sizes greater than 10-15 mm in diameter. When tumors are of this size or smaller, their MAF is about 0.01% (one tumor DNA molecule admixed with 10,000 normal DNA molecules). The use of 10 mL of blood (4 mL of plasma) will likely contain less than a complete cancer genome, thus rendering the diagnosis of cancer impossible. Grail's new data confirm the low sensitivity for early cancer detection (<30% for Stage I-II tumors, <20% for Stage I tumors), but specificity was high at 99.5%. According to these latest data, the sensitivity of the Grail test is less than 20% in Stage I disease, casting doubt if this test could become a viable pan-cancer clinical screening tool.

Cardiac troponin and COVID-19 severity: Results from BIOCOVID study.

BACKGROUND: Myocardial injury is a common finding in COVID-19 strongly associated with severity. We analysed the prevalence and prognostic utility of myocardial injury, characterized by elevated cardiac troponin, in a large population of COVID-19 patients, and further evaluated separately the role of troponin T and I. METHODS: This is a multicentre, retrospective observational study enrolling patients with laboratory-confirmed COVID-19 who were hospitalized in 32 Spanish hospitals. Elevated troponin levels were defined as values above the sex-specific 99th percentile upper reference limit, as recommended by international guidelines. Thirty-day mortality was defined as endpoint. RESULTS: A total of 1280 COVID-19 patients were included in this study, of whom 187 (14.6%) died during the hospitalization. Using a nonspecific sex cut-off, elevated troponin levels were found in 344 patients (26.9%), increasing to 384 (30.0%) when a sex-specific cut-off was used. This prevalence was significantly higher (42.9% vs 21.9%; P < .001) in patients in whom troponin T was measured in comparison with troponin I. Sex-specific elevated troponin levels were significantly associated with 30-day mortality, with adjusted odds ratios (ORs) of 3.00 for total population, 3.20 for cardiac troponin T and 3.69 for cardiac troponin I. CONCLUSION: In this multicentre study, myocardial injury was a common finding in COVID-19 patients. Its prevalence increased when a sex-specific cut-off and cardiac troponin T were used. Elevated troponin was an independent predictor of 30-day mortality, irrespective of cardiac troponin assay and cut-offs to detect myocardial injury. Hence, the early measurement of cardiac troponin may be useful for risk stratification in COVID-19.

Characteristics and laboratory findings on admission to the emergency department among 2873 hospitalized patients with COVID-19: the impact of adjusted laboratory tests in multicenter studies. A multicenter study in Spain (BIOCOVID-Spain study).

Identification of predictors for severe disease progression is key for risk stratification in COVID-19 patients. We aimed to describe the main characteristics and identify the early predictors for severe outcomes among hospitalized patients with COVID-19 in Spain. This was an observational, retrospective cohort study (BIOCOVID-Spain study) including COVID-19 patients admitted to 32 Spanish hospitals. Demographics, comorbidities and laboratory tests were collected. Outcome was in-hospital mortality. For analysis, laboratory tests values were previously adjusted to assure the comparability of results among participants. Cox regression was performed to identify predictors. Study population included 2873 hospitalized COVID-19 patients. Nine variables were independent predictors for in-hospital mortality, including creatinine (Hazard ratio [HR]:1.327; 95% Confidence Interval [CI]: 1.040-1.695, p = .023), troponin (HR: 2.150; 95% CI: 1.155-4.001; p = .016), platelet count (HR: 0.994; 95% CI: 0.989-0.998; p = .004) and C-reactive protein (HR: 1.037; 95% CI: 1.006-1.068; p = .019). This is the first multicenter study in which an effort was carried out to adjust the results of laboratory tests measured with different methodologies to guarantee their comparability. We reported a comprehensive information about characteristics in a large cohort of hospitalized COVID-19 patients, focusing on the analytical features. Our findings may help to identify patients early at a higher risk for an adverse outcome.