An LC-MS/MS-based method for the simultaneous quantification of melatonin, cortisol and cortisone in saliva.

Disturbances in the diurnal pattern are associated with several clinical and psychological conditions, including depression and fatigue. Salivary sampling for melatonin, cortisol and cortisone provides a non-invasive method for frequent sampling and obtaining biochemical insight into the diurnal pattern of individuals. Therefore, a new liquid chromatography-tandem mass spectrometry-based method for the measurement of salivary melatonin, cortisol and cortisone was developed and validated. The method required 250 µl saliva, used isotope dilution methodology and was based on a liquid-liquid extraction for sample preparation, reversed-phase chromatography and multiple reaction monitoring on a mass spectrometer for quantitation. The lower limits of quantification obtained were 0.010 nmol/L for melatonin, 0.5 nmol/L for cortisol and 1.00 nmol/L for cortisone and the limits of detection were 0.003 nmol/L, 0.15 nmol/L and 0.1 nmol/L respectively. The method imprecision was ≤14% for all measurands, and the method comparison showed highly comparable results with high correlation coefficients (all ≥0.964). Potential interference of cortisol and cortisone by prednisolone was observed and could be detected by chromatogram review. Typical diurnal patterns for melatonin, cortisol and cortisone were observed in the saliva of 20 cancer survivors who collected saliva throughout the day.

An Automated Correction Algorithm (ALPACA) for ddPCR Data Using Adaptive Limit of Blank and Correction of False Positive Events Improves Specificity of Mutation Detection.

BACKGROUND: Bio-Rad droplet-digital PCR is a highly sensitive method that can be used to detect tumor mutations in circulating cell-free DNA (cfDNA) of patients with cancer. Correct interpretation of ddPCR results is important for optimal sensitivity and specificity. Despite its widespread use, no standardized method to interpret ddPCR data is available, nor have technical artifacts affecting ddPCR results been widely studied. METHODS: False positive rates were determined for 6 ddPCR assays at variable amounts of input DNA, revealing polymerase induced false positive events (PIFs) and other false positives. An in silico correction algorithm, known as the adaptive LoB and PIFs: an automated correction algorithm (ALPACA), was developed to remove PIFs and apply an adaptive limit of blank (LoB) to individual samples. Performance of ALPACA was compared to a standard strategy (no PIF correction and static LoB = 3) using data from commercial reference DNA, healthy volunteer cfDNA, and cfDNA from a real-life cohort of 209 patients with stage IV nonsmall cell lung cancer (NSCLC) whose tumor and cfDNA had been molecularly profiled. RESULTS: Applying ALPACA reduced false positive results in healthy cfDNA compared to the standard strategy (specificity 98 vs 88%, P = 10-5) and stage IV NSCLC patient cfDNA (99 vs 93%, P = 10-11), while not affecting sensitivity in commercial reference DNA (70 vs 68% P = 0.77) or patient cfDNA (82 vs 88%, P = 0.13). Overall accuracy in patient samples was improved (98 vs 92%, P = 10-7). CONCLUSIONS: Correction of PIFs and application of an adaptive LoB increases specificity without a loss of sensitivity in ddPCR, leading to a higher accuracy in a real-life cohort of patients with stage IV NSCLC.

Validation of a clinical blood-based decision aid to guide immunotherapy treatment in patients with non-small cell lung cancer.

BACKGROUND: The widespread introduction of immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) has led to durable responses but still many patients fail and are treated beyond progression. OBJECTIVE: This study investigated whether readily available blood-based tumor biomarkers allow accurate detection of early non-responsiveness, allowing a timely switch of therapy and cost reduction. METHODS: In a prospective, observational study in patients with NSCLC treated with nivolumab or pembrolizumab, five serum tumor markers were measured at baseline and every other week. Six months disease control as determined by RECIST was used as a measure of clinical response. Patients with a disease control <  6 months were deemed non-responsive. For every separate tumor marker a criterion for predicting of non-response was developed. Each marker test was defined as positive (predictive of non-response) if the value of that tumor marker increased at least 50% from the value at baseline and above a marker dependent minimum value to be determined. Also, tests based on combination of multiple markers were designed. Specificity and sensitivity for predicting non-response was calculated and results were validated in an independent cohort. The target specificity of the test for detecting non-response was set at >  95%, in order to allow its safe use for treatment decisions. RESULTS: A total of 376 patients (training cohort: 180, validation cohort: 196) were included in our analysis. Results for the specificity of the single marker tests in the validation set were CEA: 98·3% (95% CI: 90·9-100%), NSE: 96·5% (95% CI: 87·9-99·6%), SCC: 96·5% (95% CI: 88·1-99·6%), Cyfra21·1 : 91.8% (95% CI: 81·9-97·3%), and CA125 : 86·0% (95% CI: 74·2-93·7%). A test based on the combination of Cyfra21.1, CEA and NSE accurately predicted non-response in 32.3% (95% CI 22.6-43.1%) of patients 6 weeks after start of immunotherapy. Survival analysis showed a significant difference between predicted responders (Median PFS: 237 days (95% CI 184-289 days)) and non-responders (Median PFS: 58 days (95% CI 46-70 days)) (p <  0.001). CONCLUSIONS: Serum tumor marker based tests can be used for accurate detection of non-response in NSCLC, thereby allowing early and safe discontinuation of immunotherapy in a significant subset of patients.

Serum testosterone measured by liquid chromatography-tandem mass spectrometry is an independent predictor of response to castration in metastatic hormone-sensitive prostate cancer.

BACKGROUND: Although testosterone levels have been associated with progression-free survival (PFS) in metastatic hormone-sensitive prostate cancer (mHSPC) patients, this has primarily been investigated using inaccurate immunoassays (IA). Here, we investigated whether castrate testosterone levels determined by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay is an independent risk factor for treatment response in mHSPC. METHODS: In total, 106 mHSPC patients treated with luteinizing-hormone releasing-hormone (LHRH) agonists were retrospectively analyzed between March 2018 and August 2021. Testosterone levels in serum samples were quantitated using an LC-MS/MS assay. In a subset of patients, IA (Roche Cobas Pro) values were compared with LC-MS/MS results. Survival analysis was performed using Kaplan-Meier curves and Cox proportional hazard models. RESULTS: Median PFS was shorter for high testosterone levels (>0.231 nmol/L, 18.4 v. 42.6 months, HR 1.7, p = 0.018). Low testosterone levels and a PSA response below 4 ng/mL was associated with longer median PFS (46.2 months) than the remaining combinations (13.8-19.3 months, 3.4-5.8, overall p < 0.01). In 67 patients, testosterone levels below the median remained associated with longer PFS, whereas IA measurements did not show a similar difference. CONCLUSION: Our results suggest that high castration testosterone levels measured by LC-MS/MS is an independent response predictor for mHSPC patients.

Predictive value of low testosterone concentrations during and prior to enzalutamide treatment in metastatic castration-resistant prostate cancer.

BACKGROUND: Enzalutamide is an effective treatment for metastatic castration-resistant prostate cancer (mCRPC) patients. However, variances in responses are observed and there is a need for biomarkers predicting treatment outcome and selection. In this study, we aimed to explore the predictive value of testosterone for first-line enzalutamide treatment of mCRPC. METHODS: A retrospective analysis of 72 mCRPC patients with no prior abiraterone or docetaxel treatment was performed. Serum testosterone was measured using a liquid chromatography tandem-mass spectrometry method. Association of pre- and during-enzalutimide treatment testosterone levels with progression-free survival (PFS) and failure-free survival (FFS) was investigated using univariate and multivariate Cox models. Testosterone levels were dichotomized into a low (Q1) and high (interquartile range-Q4) group. RESULTS: Median PFS (7.4 v. 20.8 months, P<0.0001) and FFS (6.6 v. 17.7 months, P<0.0001) were shorter for patients with low testosterone levels (<0.217 nmol/L) during enzalutamide treatment. Furthermore, univariate Cox proportional hazards models revealed that low testosterone levels were associated with shorter PFS (HR 3.5, 95%CI 1.9-6.3; P<0.001) and FFS (HR 3.1, 95%CI 1.7-5.5; P<0.001). Pre-treatment testosterone levels were lower than during-treatment levels (P<0.0001) and low pre-treatment testosterone levels (<0.143 nmol/L) were associated with shorter median PFS (12.6 v. 20.5 months, P<0.01) and FFS (12.6 v. 22.5 months, P<0.01). CONCLUSION: The results of this study suggest that low serum testosterone levels during and prior to enzalutamide treatment can predict progression in mCRPC patients and identifies tumors resistant to next-in-line enzalutamide treatment. Validation in a prospective cohort is warranted.

A Micro-Costing Framework for Circulating Tumor DNA Testing in Dutch Clinical Practice.

Circulating tumor DNA (ctDNA) is a promising new biomarker with multiple potential applications in cancer care. Estimating total cost of ctDNA testing is necessary for reimbursement and implementation, but challenging because of variations in workflow. We aimed to develop a micro-costing framework for consistent cost calculation of ctDNA testing. First, the foundation of the framework was built, based on the complete step-wise diagnostic workflow of ctDNA testing. Second, the costing method was set up, including costs for personnel, materials, equipment, overhead, and failures. Third, the framework was evaluated by experts and applied to six case studies, including PCR-, mass spectrometry-, and next-generation sequencing-based platforms, from three Dutch hospitals. The developed ctDNA micro-costing framework includes the diagnostic workflow from blood sample collection to diagnostic test result. The framework was developed from a Dutch perspective and takes testing volume into account. An open access tool is provided to allow for laboratory-specific calculations to explore the total costs of ctDNA testing specific workflow parameters matching the setting of interest. It also allows to straightforwardly assess the impact of alternative prices or assumptions on the cost per sample by simply varying the input parameters. The case studies showed a wide range of costs, from €168 to €7638 ($199 to $9124) per sample, and generated information. These costs are sensitive to the (coverage of) platform, setting, and testing volume.

Combining variant detection and fragment length analysis improves detection of minimal residual disease in postsurgery circulating tumour DNA of stage II-IIIA NSCLC patients.

Stage II-IIIA nonsmall cell lung cancer (NSCLC) patients receive adjuvant chemotherapy after surgery as standard-of-care treatment, even though only approximately 5.8% of patients will benefit. Identifying patients with minimal residual disease (MRD) after surgery using tissue-informed testing of postoperative plasma circulating cell-free tumour DNA (ctDNA) may allow adjuvant therapy to be withheld from patients without MRD. However, the detection of MRD in the postoperative setting is challenging, and more sensitive methods are urgently needed. We developed a method that combines variant calling and a novel ctDNA fragment length analysis using hybrid capture sequencing data. Among 36 stage II-IIIA NSCLC patients, this method distinguished patients with and without recurrence of disease in a 20 times repeated 10-fold cross validation with 75% accuracy (P = 0.0029). In contrast, using only variant calling or only fragment length analysis, no signification distinction between patients was shown (P = 0.24 and P = 0.074 respectively). In addition, a variant-level fragmentation score was developed that was able to classify variants detected in plasma cfDNA into tumour-derived or white-blood-cell-derived variants with 84% accuracy. The findings in this study may help drive the integration of various types of information from the same data, eventually leading to cheaper and more sensitive techniques to be used in this challenging clinical setting.

Retrospective analysis of serum testosterone levels by LC-MS/MS in chemically castrated prostate cancer patients: Biological variation and analytical performance specifications.

BACKGROUND: A sensitive liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was used to monitor serum testosterone levels in castrated prostate cancer patients. We subsequently performed an observational and retrospective study to estimate the within- and between-subject biological variation of these patients. METHODS: In total, 474 samples from 72 prostate cancer patients in the Netherlands receiving either chemical castration (CAS) or castration plus enzalutamide (ENZA) treatment were selected for data analysis. ANOVA was performed to estimate analytical variation (CVA) and within-patient variation (CVI). A nested ANOVA was applied to estimate between-patient variation (CVG). From these data, the reference change value (RCV) and analytical performance specifications (APS) were calculated. RESULTS: Testosterone levels were significantly higher in the ENZA group (0.318 vs. 0.191 nmol/L, p < 0.005) than the CAS group. Overall, variation components were estimated at 6.1%, 24.6% and 60.3% for CVA, CVI and CVG, respectively. Both groups showed high individuality (<0.6). The RCV was 70.3% for all patients. Desirable APS were 12.3% for imprecision, 16.3% for bias and 26.4% for total error. CONCLUSION: The generated APS are valuable for sensitive testosterone assays and the high individuality indicates that castrated testosterone levels can be studied as a predictive or prognostic biomarker in prostate cancer patients.

Light Therapy for Cancer-Related Fatigue in (Non-)Hodgkin Lymphoma Survivors: Results of a Randomized Controlled Trial.

PURPOSE: To evaluate the short- and long-term effects of light therapy on fatigue (primary outcome) and sleep quality, depression, anxiety, quality of life, and circadian rhythms (secondary outcomes) in survivors of (non-)Hodgkin lymphoma presenting with chronic cancer-related fatigue. METHODS: We randomly assigned 166 survivors (mean survival 13 years) to a bright white light intervention (BWL) or dim white light comparison (DWL) group. Measurements were completed at baseline (T0), post-intervention (T1), at three (T2), and nine (T3) months follow-up. A mixed-effect modeling approach was used to compare linear and non-linear effects of time between groups. RESULTS: There were no significant differences between BWL and DWL in the reduction in fatigue over time. Both BWL and DWL significantly (p < 0.001) improved fatigue levels during the intervention followed by a slight reduction in this effect during follow-up (EST0-T1 = -0.71; EST1-T3 = 0.15). Similar results were found for depression, sleep quality, and some aspects of quality of life. Light therapy had no effect on circadian rhythms. CONCLUSIONS: BWL was not superior in reducing fatigue compared to DWL in HL and DLBCL survivors. Remarkably, the total sample showed clinically relevant and persistent improvements on fatigue not commonly seen in longitudinal observational studies in these survivors.