Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Evaluation of an indirect immunofluorescence assay for strongyloidiasis as a tool for diagnosis and follow-up.

The diagnostic accuracy of an indirect immunofluorescence antibody test (IFAT) for Strongyloides stercoralis at different serum antibody titers was evaluated. To assess diagnostic sensitivity, sera from 156 patients with known strongyloidiasis were collected. Negative control sera were obtained from a composite group of 427 subjects (blood donors and hospitalized patients). With an area under the receiver-operating characteristic plot of 0.98, the IFAT showed a high level of diagnostic accuracy for strongyloidiasis. An antibody titer of > or = 1:20, with 97% sensitivity and 98% specificity, was identified as the diagnostic threshold with the best overall performance. Cross-reactions were evaluated with 41 additional samples from patients with other known helminth infections, and the IFAT detected low-titer positivity in only one subject with filariasis. A positive IFAT result at an antibody dilution of > or = 1:80 was virtually 100% specific, with 71% sensitivity. To test the usefulness of the IFAT as a monitoring tool, the changes in specific-antibody titers after treatment in a group of 155 patients were evaluated. Seroreversion or a decrease in antibody titer of twofold or more was observed in 60% of the patients. Response to treatment was directly correlated to the initial antibody titer, and a baseline titer of > or = 1:80 was identified as the best predictor of response. In conclusion, a positive IFAT result at an antibody dilution of >/=1:20 is the optimal cutoff for screening. A titer of > or = 1:80, with virtually no false-positive result, is a reliable cutoff for a serological assessment of treatment efficacy and for inclusion in clinical trials.

Proteomics and immunomapping of reactive lymph-node and lymphoma.

In the present study we show that two-dimensional (2-D) maps together with immuno-detection allow the precise identification of important leukocyte differentiation and tumor markers (e.g., CD3 and CD5), and important cell cycle regulatory molecules such as cyclin dependent kinases, notably CDK6. In addition, the comparative evaluation of molecular expression (e.g., CD5) in maps developed with normal and lymphoma samples can provide reproducible and precise information regarding the molecular expression in different cell populations. Accordingly, we could detect a much increased level of expression of CD5 in mantle cell lymphoma, up to ten times higher than in the control. In addition, CD5 in tumor tissues seems to be microheterogeneous as compared to normal samples.