Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens

Table 3 Validation metrics of MammaTyper amplification for each different target sequence and sample type

Versant kPCR		ERBB2	ESR1	PGR	MKI67	B2M	CALM2
FFPE Master Sample	LoD [Cq]	34.36	35.54	32.33	35.66	35.80	34.78
	LoD [molecules]	10	5	87	7	7	8
	AMR [Cq]	21.05–34.36	19.13–35.54	21.13–32.33	23.97–35.66	16.78–35.80	23.90–34.78
	Efficiency [%]	106	105	106	101	100	111
	Linearity	0.999	0.999	0.995	0.999	0.999	0.998
IVT-RNA	LoD [Cq]	35.31	35.60	36.72	36.41	36.22	33.69
	LoD [molecules]	5	5	5	5	5	18
	AMR [Cq]	12.36–35.31	12.45–35.60	12.64–36.72	11.63–36.41	12.41–36.22	11.67–33.69
	Efficiency [%]	105	104	100	99	100	100
	Linearity	0.999	0.999	1.000	0.999	1.000	1.000
LightCycler 480 II		ERBB2	ESR1	PGR	MKI67	B2M	CALM2
FFPE Master Sample	LoD [Cq]	33.56	36.47	33.56	35.47	35.6	35.62
	LoD [molecules]	40	8	64	12	12	9
	AMR [Cq]	22.32–33.56	21.69–36.47	21.20–33.56	24.61–35.47	17.60–35.60	25.26–35.62
	Efficiency [%]	109	113	115	111	109	117
	Linearity	1.000	1.000	0.996	0.999	0.999	0.997
IVT-RNA	LoD [Cq]	34.64	35.21	35.44	34.91	34.97	36.51
	LoD [molecules]	18	18	18	18	18	5
	AMR [Cq]	17.34–34.64	18.52–35.21	17.56–35.44	19.27–34.91	17.9–34.97	18.87–36.51
	Efficiency [%]	106	109	101	103	107	103
	Linearity	1.000	0.999	1.000	0.999	0.999	1.000

ISSN: 1471-2407