Table 3 Limits of detection (LOD) of the Idylla GeneFusion assay for the detection of ALK, ROS1 and RET specific gene fusions, MET exon 14 skipping as well as unknown gene fusions.

From: Validation of the Idylla GeneFusion assay to detect fusions and MET exon-skipping in non-small cell lung cancers

Commercial standard	Input (ng)	Cq of the RNA controls	Cq of the DNA control	Genomic alterations		Biological interpretation	Cq	∆Cq
SeraSeq	50	32	31.8	Specific alterations	ALK	Detected	31	–
					ROS1	Detected	30.9	–
					RET	Detected	31.6	–
					MET exon 14 skipping	Detected	30.8	–
				Expression imbalance	ALK	Detected	NA	0.2
					ROS1	Indeterminate	–	–
					RET	Indeterminate	–	–
					NTRK1	Indeterminate	–	–
					NTRK2	Not detected	31.6	5.8
					NTRK3	Detected	NA	–0.3
	30	32.3	32.4	Specific alterations	ALK	Detected	31.4	–
					ROS1	Detected	31.2	–
					RET	Detected	31.9	–
					MET exon 14 skipping	Detected	31.1	–
				Expression imbalance	ALK	Detected	NA	0.7
					ROS1	Indeterminate	–	–
					RET	Not detected	NA	5.8
					NTRK1	Indeterminate	–	–
					NTRK2	Not detected	32.3	5.8
					NTRK3	Detected	NA	−0.8
	15	35.2	34.2	Specific alterations	ALK	Detected	33.1	–
					ROS1	Detected	34.2	–
					RET	Detected	34.9	–
					MET exon 14 skipping	Detected	34.1	–
				Expression imbalance	ALK	Detected	NA	−0.3
					ROS1	Indeterminate	–	–
					RET	Indeterminate	–	–
					NTRK1	Indeterminate	–	–
					NTRK2	Indeterminate	–	–
					NTRK3	Detected	NA	−0.8
	7.5	33.2	32	Specific alterations	ALK	Detected	31.9	–
					ROS1	Detected	30.6	–
					RET	Detected	32.7	–
					MET exon 14 skipping	Detected	31.8	–
				Expression imbalance	ALK	Detected	NA	0.3
					ROS1	Indeterminate	–	–
					RET	Indeterminate	–	–
					NTRK1	Indeterminate	–	–
					NTRK2	Not detected	32.8	4.3
					NTRK3	Detected	NA	−0.6
	5	35.6	34.8	Specific alterations	ALK	Detected	34.6	–
					ROS1	Detected	34.8	–
					RET	Not detected	–	–
					MET exon 14 skipping	Detected	35.1	–
				Expression imbalance	ALK	Detected	NA	0.5
					ROS1	Indeterminate	–	–
					RET	Indeterminate	–	–
					NTRK1	Indeterminate	–	–
					NTRK2	Indeterminate	–	–
					NTRK3	Detected	NA	−0.2
	3	not amplified	34.6	Invalid
Horizon	15	34.6	35	Specific alterations	ALK	Detected	34.5	–
					ROS1	Detected	34.6	–
					RET	Detected	32.2	–
					MET exon 14 skipping	Not detected	–	–
				Expression imbalance	ALK	Detected	NA	0.7
					ROS1	Detected	38.5	−5
					RET	Detected	36.3	−1.9
					NTRK1	Not detected	37.8	−1
					NTRK2	Indeterminate	–	–
					NTRK3	Indeterminate	–	–
	10	34.9	34.9	Specific alterations	ALK	Detected	35.8	–
					ROS1	Detected	35.3	–
					RET	Detected	32.3	–
					MET exon 14 skipping	Not detected	–	–
				Expression imbalance	ALK	Detected	NA	1.1
					ROS1	Detected	37.5	−3.2
					RET	Not detected	36	−1.2
					NTRK1	Indeterminate	–	–
					NTRK2	Indeterminate	–	–
					NTRK3	indeterminate	–	–
	7.5	Not amplified	35.7	Invalid

Interpretation of the Idylla results: the Idylla analysis is considered inconclusive if RNA control curves are invalid, and the DNa control Cq is determined to assess RNA degradation; specific fusion is found “detected” if the PCR curve is valid and the Cq appears within a predefined range; MET exon 14 skipping is found “detected” if the corresponding PCR curve is valid, the Cq is within a predefined range and the difference between METex14 and MET wild-type Cq (ΔCq) is below a predefined threshold; the detection of an “expression imbalance” indicates a difference between 3' end and 5' end expression levels of the gene and should reflect the presence of an unknown gene fusion that need to be confirmed by complementary techniques (IHC, FISH or NGS); the analysis of expression imbalance requires the validity of the 3' curve, otherwise the result is qualified as “indetermined”. If the 3′ and 5′ curves are valid, the expression imbalance is considered “detected” if the ΔCq value (i.e. the difference between 3′ and 5′ Cq) is below a predefined (5′ Cq value-dependent) threshold. If the 3′ curve is valid while the 5′ curve is invalid, the ΔCq value is calculated between 3′ and RNA control curves and the expression imbalance is found “detected” if the ΔCq value is below a predefined threshold
Cq cycle quantification, NA not applicable.

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Table 3 Limits of detection (LOD) of the Idylla GeneFusion assay for the detection of ALK, ROS1 and RET specific gene fusions, MET exon 14 skipping as well as unknown gene fusions.

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