Steven Kao

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

PURPOSE Lorlatinib is a potent, brain-penetrant, third-generation anaplastic lymphoma kinase (ALK)/ROS1 tyrosine kinase inhibitor (TKI) with robust clinical activity in advanced ALK-positive non–small-cell lung cancer, including in patients who have failed prior ALK TKIs. Molecular determinants of response to lorlatinib have not been established, but preclinical data suggest that ALK resistance mutations may represent a biomarker of response in previously treated patients. PATIENTS AND METHODS Baseline plasma and tumor tissue samples were collected from 198 patients with ALK-positive non–small-cell lung cancer from the registrational phase II study of lorlatinib. We analyzed plasma DNA for ALK mutations using Guardant360. Tumor tissue DNA was analyzed using an ALK mutation–focused next-generation sequencing assay. Objective response rate, duration of response, and progression-free survival were evaluated according to ALK mutation status. RESULTS Approximately one quarter of patients had ALK mutations detected by plasma or tissue genotyping. In patients with crizotinib-resistant disease, the efficacy of lorlatinib was comparable among patients with and without ALK mutations using plasma or tissue genotyping. In contrast, in patients who had failed 1 or more second-generation ALK TKIs, objective response rate was higher among patients with ALK mutations (62% v 32% [plasma]; 69% v 27% [tissue]). Progression-free survival was similar in patients with and without ALK mutations on the basis of plasma genotyping (median, 7.3 months v 5.5 months; hazard ratio, 0.81) but significantly longer in patients with ALK mutations identified by tissue genotyping (median, 11.0 months v 5.4 months; hazard ratio, 0.47). CONCLUSION In patients who have failed 1 or more second-generation ALK TKIs, lorlatinib shows greater efficacy in patients with ALK mutations compared with patients without ALK mutations. Tumor genotyping for ALK mutations after failure of a second-generation TKI may identify patients who are more likely to derive clinical benefit from lorlatinib.

PURPOSE: Asbestos-induced chronic inflammation is implicated in the pathogenesis of malignant mesothelioma (MM). We have investigated blood neutrophil-to-lymphocyte ratio (NLR), an index of systemic inflammation, as a prognostic factor in MM patients. EXPERIMENTAL DESIGN: Patients with MM who had systemic therapy at participating institutes were studied. Potential prognostic factors such as age, gender, performance status, histologic subtype, and baseline laboratory parameters, including NLR, were analyzed. Overall survival from commencement of therapy was determined by the Kaplan-Meier method. Multivariate analyses using Cox Regression model were performed with significant factors (P ≤ 0.05) to determine their independent effect. RESULTS: A total of 173 MM patients undergoing systemic therapy including 119 patients receiving first-line therapy and 54 patients receiving second- or third-line therapy were included in this retrospective evaluation. Forty-two percent of patients had an elevated NLR at baseline. The following variables were predictive of survival: female gender (P = 0.044), epithelioid histologic subtype (P < 0.001), baseline white blood cell count less than 8.3 × 10⁹/L (P = 0.008), baseline platelet count 400 × 10⁹/L or less (P = 0.05), and NLR of 5 or less (P < 0.001). After multivariate analysis, histologic epithelioid subtype [hazard ratio (HR) = 2.0; 95% confidence interval (CI) = 1.3-2.9; P = 0.001], and NLR less than 5 (HR = 2.7; 95% CI = 1.8-3.9; P < 0.001) remained independent predictors. The 1-year survival rate was 60% versus 26%, whereas the 2-year survival rate was 34% versus 10% for NLR less than 5 and 5 or greater, respectively. In the separate analyses of chemotherapy-naive and previously treated patient groups, NLR was an independent predictor of survival in both groups. CONCLUSION: Our results indicate that NLR is an independent predictor of survival for patients with MM undergoing systemic therapy.