Reflex testing in non-small cell lung carcinoma using DNA- and RNA-based next-generation sequencing—a single-center experience

Zacharias, Martin; Absenger, Gudrun; Kashofer, Karl; Wurm, Robert; Lindenmann, Jörg; Terbuch, Angelika; Konjić, Selma; Sauer, Stefan; Gollowitsch, Franz; Gorkiewicz, Gregor; Brčić, Luka

doi:10.21037/tlcr-21-570

Cited by 11 publications

(12 citation statements)

References 39 publications

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“…Nevertheless, we believe that the low frequency found in our study population reflects the real incidence in Nordic countries and, more generally, in a European population. Similar findings were reported from an independent survey of a large advanced Swedish NSCLC patient cohort that used IHC and FISH for detection ( 44 ) and from an independent study of an Austrian NSCLC patient population ( 45 ). Low frequencies have also been reported for other gene rearrangements, such as ALK, RET, and NTRK ( 8 , 46 , 47 ).…”

Section: Discussionsupporting

confidence: 84%

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas

Thurfjell¹,

Micke²,

Yu³

et al. 2022

Transl Lung Cancer Res

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Background: Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts.Methods: Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21).Results: Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between ^ ORCID: 0000-0002-5294-7808. Thurfjell et al. ROS1 in NSCLCthe assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNAsequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay. Conclusions:The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.

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Section: Discussionsupporting

confidence: 84%

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas

Thurfjell¹,

Micke²,

Yu³

et al. 2022

Transl Lung Cancer Res

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“…Currently, in many clinical laboratories, DNA‐based targeted NGS analysis has become a routine approach for mutation detection instead of multiple different traditional molecular assays, such as Sanger sequencing and qPCR. Recently, several studies demonstrated that RNA NGS was a valuable addition to identify fusions and exon‐skipping events, especially for novel or unusual gene fusions 25,28,35–37 . A study by Benayed et al.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, several studies demonstrated that RNA NGS was a valuable addition to identify fusions and exon-skipping events, especially for novel or unusual gene fusions. 25,28,[35][36][37] A study by Benayed et an RNA-based approach detected a higher proportion of MET exon 14 skipping cases than did DNA-based analysis (4.2% versus 1.3%). 17 Another study showed that treatment options were changed by adding RNA NGS analysis in 5% of all NSCLC patients, in which 22% never smoked.…”

Section: Performance Of the Drcc-seq Approach In Real-world Clinical ...mentioning

confidence: 98%

A unified DNA‐ and RNA‐based NGS strategy for the analysis of multiple types of variants at the dual nucleic acid level in solid tumors

Chen,

Wang,

Zhang

et al. 2023

Clinical Laboratory Analysis

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BackgroundTargeted next‐generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA‐based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA‐ and RNA‐based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA‐ and RNA‐based NGS approach is urgently needed in clinical practice.MethodsA DNA/RNA co‐hybrid capture sequencing (DRCC‐Seq) approach was designed to capture pre‐capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC‐Seq approach was evaluated by a panel of reference standards and clinical samples.ResultsThe average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC‐Seq approach. The DRCC‐Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real‐world clinical samples.ConclusionsThe DRCC‐Seq approach is more cost‐effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.

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“…DNA-based next-generation sequencing was performed as previously described [ 9 ]. Briefly, DNA extraction was performed for between 2 and 12 FFPE tissue sections for each case using the Maxwell 16 instrument (Promega, Mannheim, Germany) and the Maxwell RSC DNA FFPE Kit (Promega, Mannheim, Germany; CatNr: AS1450).…”

Section: Methodsmentioning

confidence: 99%

Expanding Broad Molecular Reflex Testing in Non-Small Cell Lung Cancer to Squamous Histology

Zacharias,

Konjic,

Kratochwill

et al. 2024

Cancers

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Due to the success story of biomarker-driven targeted therapy, most NSCLC guidelines agree that molecular reflex testing should be performed in all cases with non-squamous cell carcinoma (non-SCC). In contrast, testing recommendations for squamous cell carcinoma (SCC) vary considerably, specifically concerning the exclusion of patients of certain age or smoking status from molecular testing strategies. We performed a retrospective single-center study examining the value of molecular reflex testing in an unselected cohort of 316 consecutive lung SCC cases, tested by DNA- and RNA-based next-generation sequencing (NGS) at our academic institution between 2019 and 2023. Clinicopathological data from these cases were obtained from electronic medical records and correlated with sequencing results. In 21/316 (6.6%) cases, we detected an already established molecular target for an approved drug. Among these were seven cases with an EGFR mutation, seven with a KRAS G12C mutation, four with an ALK fusion, two with an EGFR fusion and one with a METex14 skipping event. All patients harboring a targetable alteration were >50 years of age and most of them had >15 pack-years, questioning restrictive molecular testing strategies. Based on our real-world data, we propose a reflex testing workflow using DNA- and RNA-based NGS that includes all newly diagnosed NSCLC cases, irrespective of histology, but also irrespective of age or smoking status.

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Reflex testing in non-small cell lung carcinoma using DNA- and RNA-based next-generation sequencing—a single-center experience

Cited by 11 publications

References 39 publications

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas

A unified DNA‐ and RNA‐based NGS strategy for the analysis of multiple types of variants at the dual nucleic acid level in solid tumors

Expanding Broad Molecular Reflex Testing in Non-Small Cell Lung Cancer to Squamous Histology

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