The transcriptomic expression pattern of immune checkpoints shows heterogeneity between and within cancer types.

Transcriptomic expression profiles of immune checkpoint markers are of interest in order to decipher the mechanisms of immunotherapy response and resistance. Overall, 514 patients with various solid tumors were retrospectively analyzed in this study. The RNA expression levels of tumor checkpoint markers (ADORA2A, BTLA, CD276, CTLA4, IDO1, IDO2, LAG3, NOS2, PD-1, PD-L1, PD-L2, PVR, TIGIT, TIM3, VISTA, and VTCN) were ranked from 0-100 percentile based on a reference population. The expression of each checkpoint was correlated with cancer type, microsatellite instability (MSI), tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) by immunohistochemistry (IHC). The cohort included 30 different tumor types, with colorectal cancer being the most common (27%). When RNA percentile rank values were categorized as "Low" (0-24), "Intermediate" (25-74), and "High" (75-100), each patient had a distinctive portfolio of the categorical expression of 16 checkpoint markers. Association between some checkpoint markers and cancer types were observed; NOS2 showed significantly higher expression in colorectal and stomach cancer (P < 0.001). Principal component analysis demonstrated no clear association between combined RNA expression patterns of 16 checkpoint markers and cancer types, TMB, MSI or PD-L1 IHC. Immune checkpoint RNA expression varies from patient to patient, both within and between tumor types, though colorectal and stomach cancer showed the highest levels of NOS2, a mediator of inflammation and immunosuppression. There were no specific combined expression patterns correlated with MSI, TMB or PD-L1 IHC. Next generation immunotherapy trials may benefit from individual analysis of patient tumors as selection criteria for specific immunomodulatory approaches.

T-cell priming transcriptomic markers: implications of immunome heterogeneity for precision immunotherapy.

Immune checkpoint blockade is effective for only a subset of cancers. Targeting T-cell priming markers (TPMs) may enhance activity, but proper application of these agents in the clinic is challenging due to immune complexity and heterogeneity. We interrogated transcriptomics of 15 TPMs (CD137, CD27, CD28, CD80, CD86, CD40, CD40LG, GITR, ICOS, ICOSLG, OX40, OX40LG, GZMB, IFNG, and TBX21) in a pan-cancer cohort (N = 514 patients, 30 types of cancer). TPM expression was analyzed for correlation with histological type, microsatellite instability high (MSI-H), tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) expression. Among 514 patients, the most common histological types were colorectal (27%), pancreatic (11%), and breast cancer (10%). No statistically significant association between histological type and TPM expression was seen. In contrast, expression of GZMB (granzyme B, a serine protease stored in activated T and NK cells that induces cancer cell apoptosis) and IFNG (activates cytotoxic T cells) were significantly higher in tumors with MSI-H, TMB ≥ 10 mutations/mb and PD-L1 ≥ 1%. PD-L1 ≥ 1% was also associated with significantly higher CD137, GITR, and ICOS expression. Patients' tumors were classified into "Hot", "Mixed", or "Cold" clusters based on TPM expression using hierarchical clustering. The cold cluster showed a significantly lower proportion of tumors with PD-L1 ≥ 1%. Overall, 502 patients (98%) had individually distinct patterns of TPM expression. Diverse expression patterns of TPMs independent of histological type but correlating with other immunotherapy biomarkers (PD-L1 ≥ 1%, MSI-H and TMB ≥ 10 mutations/mb) were observed. Individualized selection of patients based on TPM immunomic profiles may potentially help with immunotherapy optimization.

Tumor Infiltrating Lymphocyte Expression of PD-1 Predicts Response to Anti-PD-1/PD-L1 Immunotherapy.

Introduction: Many studies have focused on the role of programmed death receptor ligand 1 (PD-L1) expression in predicting immunotherapy outcomes. Limited clinical data are available regarding the role of programmed death receptor 1 (PD-1; the PD-L1 receptor) expressing tumor-infiltrating lymphocytes (TILs) in PD-1/PD-L1 antibody responsiveness. However, preclinical studies demonstrate that TILs expressing PD-1 contribute to tumor immune evasion. Methods: This study analyzed the association between TIL-PD-1 status and outcome after immune checkpoint blockade (ICB) therapy. We evaluated 123 patients with various solid tumors treated with monoclonal antibodies targeting the PD-1/PD-L1 signaling axis. Additionally, 8706 solid tumor specimens were assessed for TIL-PD-1 and tumor mutational burden (TMB) status. Results: The presence of PD-1-expressing TILs in tumors was associated with increased median progression-free survival (7.0 vs 1.9 months; p = 0.006) and overall survival (18.1 vs 8.0 months; p = 0.04) after treatment with ICB. TIL-PD-1-positive patients had an objective response rate (ORR) of 41% (95% CI, 24-61; N = 12/29) compared with 17% (95% CI, 4-43; N = 3/17) for TIL-PD-1-negative patients (p = 0.18). Analyzed as continuous variables, TIL-PD-1 and TMB showed a weak correlation in 8706 solid tumor samples (Pearson r = 0.074); when analyzed as categorical variables (cutoffs: TIL-PD-1 ≥ 1% and TMB ≥ 10 mutations/Mb), the two variables are correlated (p < 0.0001). TIL-PD-1-positive status is also associated with enrichment of pathologic variants within several genes, most notably TP53 (adjusted p < 0.05). Conclusion: TIL-PD-1 positivity in tumors (≥ 1%) is associated with significantly longer progression-free and overall survival after ICB. ClinicalTrials.gov ID: NCT02478931.

Mass Spectrometry-Based Detection of Beta Lactam Hydrolysis Enables Rapid Detection of Beta Lactamase Mediated Antibiotic Resistance.

OBJECTIVE: Antibiotic resistance by beta lactamase expression is a serious and growing threat. We aimed to determine whether beta-lactamase activity is detectable in urine specimens to enable faster identification of resistance. METHODS: Urine specimens from patients with extended spectrum beta lactamase (ESBL)-expressing urinary infections were incubated with beta lactam antibiotics. Beta lactam hydrolysis was determined by mass spectrometry methods. RESULTS: Ceftriaxone hydrolysis was observed in 45 of 45 ESBL-containing specimens from patients not treated with a beta lactamase inhibitor before specimen collection. Ceftriaxone hydrolysis was not observed in 108 of 108 non-ESBL-containing specimens. Spiking studies show that beta lactam hydrolysis can be observed within 30 minutes. Beta lactam hydrolysis is evidenced by mass spectrometry preceded by either liquid chromatography or matrix-assisted laser desorption ionization specimen processing methods. CONCLUSION: Clinically significant beta lactamase activity is detectable directly from urine specimens. The described methods would enable the detection of beta lactam resistance 24 to 48 hours sooner than culture based methods.

Emergency Department Management of Chest Pain With a High-Sensitivity Troponin-Enabled 0/1-Hour Rule-Out Algorithm.

OBJECTIVES: The analytical sensitivity of high-sensitivity cardiac troponin T (hsTnT) assays has enabled rapid myocardial infarction rule-out algorithms for emergency department (ED) presentations. Few studies have analyzed the real-world impact of hsTnT algorithms on outcomes and operations. METHODS: Comparison of ED length of stay (LOS) and 30-day outcomes (return to ED, inpatient admission, and mortality) for patients presenting with chest pain during 2 separate 208-day periods using a 0/1-hour hsTnT-enabled algorithm or fourth-generation TnT. RESULTS: Discharge, 30-day readmission, and 30-day mortality rates were not significantly different with fourth-generation TnT vs hsTnT. Thirty-day return rates were significantly decreased with hsTnT (17.4% vs 14.9%; P < .01). For encounters with TnT measured at least twice and resulting in discharge, median ED LOS decreased by 61 minutes with the use of hsTnT (488 vs 427 minutes; P < .0001). Median time between first and second TnT results decreased by 82 minutes with hsTnT (202 vs 120 minutes; P < .0001), suggesting that the 0/1-hour algorithm was incompletely adopted. CONCLUSIONS: Implementation of the hsTnT algorithm was associated with decreased 30-day return rates and decreased ED LOS for a subset of patients, despite incomplete adoption of the 0/1-hour algorithm.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

BACKGROUND: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. METHODS: The ability of 4 commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 patients who were SARS-CoV-2 PCR positive, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 patients who were SARS-CoV-2 negative. Serology results were compared to a cell-based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. RESULTS: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received 2 doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme NAbs assay correlated with the PSV SARS-CoV-2 median infective dose (ID50) neutralization titers (R2 = 0.70), while correlation of the Roche S-antibody assay was weaker (R2 = 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received 2 doses of the Moderna vaccine (ID50, 597) compared to individuals who received a single dose (ID50, 284). CONCLUSIONS: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

Test Volume Ratio Benchmarking to Identify and Reduce Low-Value Laboratory Utilization.

OBJECTIVES: We analyzed test volume data to identify low-value test utilization. We subsequently tracked the efficacy of interventions to improve test utilization by decreasing low-value testing. METHODS: Test volume data for analytes included in the Choosing Wisely guidelines were analyzed to identify population outliers. Outliers were defined by test volume ratios of either analyte to sodium or paired analytes to correct for variation in patient volumes at each site. Interventions to improve test utilization were targeted to outlier sites. Relative efficacy in reducing low-value testing was tracked at those sites. RESULTS: After appropriate data cleaning, test volume ratios for 17 analytes paired with sodium and 8 pairs of analytes were acquired from 108 national sites. A site with abnormally high Clostridium difficile/sodium ratio was selected for intervention, leading to a 71% decrease in C difficile tests. Two different interventions to decrease creatine kinase MB isoform (CKMB) testing were performed at two unique sites with abnormally high CKMB/troponin ratios. These interventions decreased CKMB by 11% and 98% at the different sites, showing the efficacy of the different kinds of interventions. CONCLUSIONS: Test volume ratio analysis and benchmarking enable identification of low-value test utilization.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

Background: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. Methods: The ability of four commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 SARS-CoV-2 PCR-positive patients, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 SARS-CoV-2 negative patients. Serology results were compared to a cell based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. Results: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received two-doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme Nabs assay correlated with the PSV SARS-CoV-2 ID50 neutralization titers (R2= 0.70), while correlation of the Roche S-antibody assay was weaker (R2= 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received two-doses of the Moderna vaccine (ID50: 597) compared to individuals that received a single dose (ID50: 284). Conclusions: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

Calibrating from Within: Multipoint Internal Calibration of a Quantitative Mass Spectrometric Assay of Serum Methotrexate.

BACKGROUND: Clinical LC-MS/MS assays traditionally require that samples be run in batches with calibration curves in each batch. This approach is inefficient and presents a barrier to random access analysis. We developed an alternative approach called multipoint internal calibration (MPIC) that eliminated the need for batch-mode analysis. METHODS: The new approach used 4 variants of 13C-labeled methotrexate (0.026-10.3 µM) as an internal calibration curve within each sample. One site carried out a comprehensive validation, which included an evaluation of interferences and matrix effects, lower limit of quantification (LLOQ), and 20-day precision. Three sites evaluated assay precision and linearity. MPIC was also compared with traditional LC-MS/MS and an immunoassay. RESULTS: Recovery of spiked analyte was 93%-102%. The LLOQ was validated to be 0.017 µM. Total variability, determined in a 20-day experiment, was 11.5%CV. In a 5-day variability study performed at each site, total imprecision was 3.4 to 16.8%CV. Linearity was validated throughout the calibrator range (r2 > 0.995, slopes = 0.996-1.01). In comparing 40 samples run in each laboratory, the median interlaboratory imprecision was 6.55%CV. MPIC quantification was comparable to both traditional LC-MS/MS and immunoassay (r2 = 0.96-0.98, slopes = 1.04-1.06). Bland-Altman analysis of all comparisons showed biases rarely exceeding 20% when MTX concentrations were >0.4 µM. CONCLUSION: The MPIC method for serum methotrexate quantification was validated in a multisite proof-of-concept study and represents a big step toward random-access LC-MS/MS analysis, which could change the paradigm of mass spectrometry in the clinical laboratory.

A Dilution Method to Mitigate Biotin Interference in Cardiac Troponin T Testing.

BACKGROUND: Oral biotin supplementation is known to interfere with biotin-streptavidin-based immunoassays, including Roche's fifth-generation cardiac troponin T (cTnT) assay, which plays a critical role in the diagnosis of myocardial infarction (MI). The utility of dilution, a quick and easy method to detect and remove interferences, has not been published for biotin interference. METHODS: Concentrations of cTnT were measured in pooled serum from clinical samples. Serum samples were supplemented with biotin to known concentrations, then cTnT concentrations were remeasured to assess for biotin interference. Samples were then diluted to assess for effective removal of biotin interference. RESULTS: At cTnT values near the critical reporting range for our institution (100 ng/L) we observed significant interference in measured values with added biotin concentrations above 50 ng/mL. In specimens without added biotin, autodilution at a 1:10 ratio yielded a mean 157% capture of measured cTnT, precluding the use of autodilution for detecting and mitigating biotin interference. A 1:10 dilution with serum containing 20-30 ng/L cTnT yielded a mean capture of 107%, which was suitable for detecting underlying biotin interference in supplemented samples. CONCLUSIONS: Biotin interference, at supraphysiologic concentrations, may create an artifactual reduction in measured cTnT to levels that could lead to delayed detection of an MI. Dilution with serum of known cTnT concentration of 20-30 ng/L is a fast and effective method to mitigate the analytical consequences of biotin interference.

Comparison of Different Small Clinical Hematology Laboratory Configurations With Focus on Remote Smear Imaging.

CONTEXT.­: Stand-alone clinical sites (eg, infusion centers) are becoming increasingly common. These sites require timely hematology analysis. Here we compare performance and costs of currently available analysis configurations with special focus on a proposed alternative using a minimal hematology analyzer plus a digital imaging device, allowing for remote oversight and interpretation. OBJECTIVES.­: To determine whether low-volume laboratories might realize savings while gaining function by substituting commonly used configurations with a proposed alternative. DESIGN.­: To evaluate the performance of the proposed alternative configuration, blood counts with automated differentials produced by a Sysmex XE5000 (complete blood count reference method) were compared with cell counts from the Sysmex pocH-100i, CellaVision DM96 preclassified differentials, and DM96 reclassified differentials (differential reference method) by using standard regression analyses, 95% CIs, and truth tables. Financial cost modeling used staffing practices, test volumes, and smear production rates observed at remote clinics performing on-site hematology analysis within the University of California at San Diego Health system. RESULTS.­: Differential blood count parameters showed excellent correlation between the XE5000 and preclassification DM96 with R2 > 0.95. For blasts/abnormal cells, immature granulocytes, and nucleated red blood cells, the DM96 showed higher sensitivity and similar specificity to the XE5000. Cost modeling revealed that decreased personnel costs through remote monitoring of results facilitated by the DM96 would lead to lower operational costs relative to more conventional analysis configurations. CONCLUSIONS.­: A digital imaging instrument with an inexpensive hematology analyzer provides similar information to a complex hematology analyzer and allows remote review of the blood smear findings by experts, leading to significant cost savings.