ALK fusion variants detection by targeted RNA-next generation sequencing and clinical responses to crizotinib in ALK-positive non-small cell lung cancer.

OBJECTIVES: The aim of the present study was firstly to assess in a clinical setting the yields of an amplicon-based parallel RNA sequencing (RNA-seq) assay for ALK fusion transcript variants detection in comparison with immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) in a selected population of ALK-positive and ALK-negative non-small cell lung cancer (NSCLC) cases, and secondly to evaluate the impact of the ALK variant on crizotinib efficacy. MATERIALS AND METHODS: The cohort used for the assessment of the RNA-seq assay comprised 53 samples initially diagnosed as being ALK-positive based on the results obtained by IHC and/or FISH, and 23 ALK-negative samples. A distinction was made between 'truly' IHC/FISH positive or 'truly' IHC/FISH negative samples, and those for which the IHC and/or FISH were equivocal (IHC) or borderline-positive (FISH). RESULTS: On the overall population, RNA-seq sensitivity (Se) and specificity (Spe) were of 80% and 100%, respectively when IHC and FISH were combined. For the 31 'truly positive' samples, Se and Spe of 100% were reached. An ALK status could be assigned by RNA-seq in 10/10 of the equivocal and/or borderline-positive IHC/FISH cases, 2/7 IHC/FISH discordant cases. When crizotinib efficacy was evaluated according to the type of ALK variant, better clinical outcomes were observed in crizotinib-treated patients with EML4-ALK v1/v2/others variants compared to v3a/b variants. CONCLUSION: RNA-seq detects ALK rearrangements with a high sensitivity and specificity using only 10 ng of RNA. It appears to be a promising rescue technique for non-clear-cut IHC/FISH cases and also offers a unique opportunity to identify ALK fusion variants and evaluate their predictive value for ALK inhibitors efficacy.

Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood.

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.