Table 2: Analytical methods validation.

Biomarkers (alphabetically)	Assay	Origin	Validation parameters										Drug	Phase	Year	Ref #
Biomarkers (alphabetically)	Assay	Origin	I	II	III	IV	V	VI	VII	VIII	IX	X	Drug	Phase	Year	Ref #
ADA	ELISA	LDA	+	+	+	+	+	+	+	+/+	+	+	Blinatumomab	1	2018	34
ADA	LBA	Comm.	+	+	+	+	+	-	+	+/+	+	+	Regorafenib	1	2016	13
ADA	ELISA	LDA	-	-	-	-	-	+	-	-/-	-	-	TAS-266	1	2015	75
ADA	ELISA	LDA	-	-	-	-	-	+	-	-/-	-	-	Angiocal	1	2013	67
Ang2	ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	+	Docetaxel	2	2014	52
B/T-cells	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	Birinapant	2	2016	86
BTK	LBA	LDA	+	-	+	+	-	+	+	+/+	-	+	Tirabrutinib	1	2018	26
BTK	ELISA	LDA	+	-	-	-	-	+	-	-/-	-	-	Acalabrutinib	1	2018	25
ccK18	ELISA	Comm.	+	+	+	+	+	+	-	+/+	+	+	SOR-C13	1	2017	51
CD137	xMAP ELISA	Comm.	+	-	+	+	+	+	+	+/+	+	+	Utomilumab	1	2018	56
CD26	FC	LDA	+	-	-	-	-	-	-	-/-	-	-	YS-110	1	2017	58
CD3/14/20 cells	FC	LDA	-	-	-	+	-	-	-	-/-	-	-	CC-223	1	2015	45
CEC	FC	LDA	+	-	+	-	-	-	-	+/-	-	-	Pazopanib	1	2014	36
CgA	RIA	Comm.	-	-	-	-	-	-	+	+/+	-	-	Lanreotide	3	2016	31
Collagen IV	ELISA	Comm.	+	-	-	+	-	+	-	-/-	-	-	Cediranib	1	2015	35
CRP	LBA	n.d.	-	-	-	-	-	-	+	-/-	-	-	Siltuximab	1	2013	21
CTC	FC	Comm.	+	-	+	+	+	+	+	+/+	+	+	ASP-9521	1/2	2014	85
CTC	FC	Comm.	+	-	+	+	+	+	+	+/+	+	+	ARN-509	1	2013	44
CTC	FC	LDA	-	-	-	-	-	+	-	-/-	-	-	Alisertib	1/2	2017	79
CTC	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	TRC105	2	2016	61
mCTC	FC	LDA	+	+	+	+	+	+	+	+/+	+	+	5‑F‑2′‑dC	1	2020	24
CTX1	ELISA	Comm.	+	-	-	+	-	+	+	+/+	+	-	GLPG-0187	1	2016	17
Cytokines	xMAP ELISA	Comm.	+	-	+	+	+	+	+	+/+	+	+	TMX-101	1	2015	10
dFdC-TP	LC-MS/MS	LDA	+	-	+	+	+	+	+	+/+	+	+	Gemcitabine	1/2	2015	38
DNA	LC-MS/MS	LDA	+	+	+	+	+	+	+	+/+	+	+	LY2603618	2	2017	62
EGF	LBA	LDA	-	-	-	-	-	-	-	-/-	-	-	Sorafenib	1	2015	37
EPO	LBA	Comm.	+	+	+	+	-	+	-	+/+	+	-	PT2385	1	2018	66
FGF	ELISA	Comm.	+	-	-	+	-	-	+	+/+	-	-	S49076	1	2017	70
FGFR	ELISA	LDA	-	-	-	-	-	-	-	-/-	-	-	JNJ-42756493	1	2015	71
G-CSF	ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	-	Lenvatinib	1	2013	43
GSH-GSSH	Spectrometry	Comm.	-	-	-	-	-	+	-	-/-	-	-	IACS-010759	1	2020	30
γ-H2AX	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	MK-8776	1	2015	42
HSP70	ELISA	n.d.	-	-	-	-	-	-	-	-/-	-	-	NVP-AUY922	2	2014	77
γ-IF	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	Cixutumumab	2	2014	12
IGF1	ELISA	Comm.	+	-	+	+	-	+	-	+/+	+	-	Polyphenon E	2	2017	39
IGF1	ELISA	Comm.	+	-	+	+	-	-	+	+/+	+	-	MEDI-573	1	2015	54
IGF1R	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	BIIB-022	1	2014	72
IGFBP2	ELISA	Comm.	+	+	+	+	+	+	-	+/+	+	-	CNF-1010	1	2013	65
Il-2	ELISA	n.d.	-	-	-	-	-	-	-	-/-	-	-	RO4929097	2	2015	29
Il2-10	ELISA	Comm.	-	-	-	-	-	-	-	-/-	-	-	Ponatinib	2	2019	74
Il-8	ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	+	Sunitinib	3	2013	87
LFA102	ELISA	LDA	-	-	-	+	-	-	-	-/-	-	-	LFA-102	1	2016	83
lymphocytes	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	Dinaciclib	1	2013	33
monocytes	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	Emactuzumab	1	2019	19
p-AKT	ELISA	Comm.	-	+	+	+	+	+	-	+/+	+	-	PX-866	2	2019	82
p-AKT	xMAP ELISA	Comm.	+	-	+	+	-	+	-	+/+	-	-	Pictilisib	1	2016	41
p-AKT	xMAP ELISA	Comm.	+	-	+	+	+	+	+	+/+	+	+	Pictilisib	1	2015	59
PAR	ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	+	Olaparib	1	2018	16
PAR	ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	+	ABT-888	1	2017	57
p-BAD	ELISA	Comm.	+	-	-	+	-	+	-	+/-	-	-	AZD1208	1	2018	46
p-CREB	FC	LDA	+	-	-	-	-	-	-	+/-	-	-	AB-928	1	2018	80
PD1	ELISA	LDA	+	+	+	+	+	+	+	+/+	+	+	SHR-1210	1	2018	18
PDGF	LBA	Comm.	+	-	-	+	+	-	+	+/+	+	-	Motesanib	2	2015	48
PDGF	xMAP ELISA	Comm.	+	-	+	+	+	+	+	+/+	+	+	MEDI‑575	1	2014	40
PDGF	ELISA	Comm.	+	+	+	+	+	+	-	+/+	+	-	Pazopanib	1	2013	47
PD-L1	FC	LDA	+	-	-	-	-	-	-	-/-	-	-	Velumab	1a	2017	53
p-ERK	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	TAK-733	1	2016	73
p-FAK	ELISA	Comm.	+	-	+	+	+	+	-	+/+	+	-	BI 853520	1	2019	60
PI3K	FC	LDA	-	-	-	-	-	-	-	-/-	-	-	Temsirolimus	1	2017	78
p-RAD50	MRM-MS	LDA	+	-	+	+	+	+	+	+/+	+	+	AZD0156	1	2018	14
PRAS40	ELISA	Comm.	+	-	+	+	+	+	-	+/+	+	-	Azacitidine	1	2017	32
p-SCR	ELISA	Comm.	+	-	-	-	-	-	-	-/-	-	-	Dasatinib	2	2019	23
Rhodamine	Spectrometry	LDA	+	-	+	-	-	-	-	+/+	-	-	Tariquidar	1	2015	68
RON8	ELISA	Comm.	+	-	-	+	-	-	-	-/-	-	-	Narnatumab	1	2017	55
Serum IgG	xMAP ELISA	Comm.	+	-	+	+	+	+	+	+/+	+	+	Sipuleucel-T	3	2015	15
serum proteins	xMAP ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	+	Cediranib	3	2014	84
s-VEGF	ELISA	Comm.	+	-	+	+	-	+	+	+/+	+	-	Bevacizumab	2	2014	81
s-VEGF1/2	ELISA	n.d.	-	-	-	-	-	-	-	-/-	-	-	Lenvatinib	1	2014	69
s-VEGF1/2	ELISA	Comm.	+	+	+	+	+	+	+	+/+	+	+	Ramucirumab	3	2020	22
s-VEGFR2	ELISA	n.d.	-	-	-	-	-	-	-	-/-	-	-	XL-647	1	2018	49
Testosterone	HPLC	LDA	+	-	+	+	+	+	+	+/+	+	+	Abiraterone	2	2018	64
Testosterone	LC-MS/MS	LDA	+	+	+	+	+	+	+	+/+	+	+	Relugolix	1	2015	11
Testosterone	LC-MS/MS	LDA	-	-	-	-	-	-	+	-/-	-	-	Orteronel	1	2015	27
TSA	radio-assay	LDA	+	+	+	+	+	+	+	+/+	+	+	Capecitabine	1	2020	63
VEGF	ELISA	Comm.	+	-	+	+	-	+	+	+/+	+	+	Cediranib	1	2015	76
VEGF	ELISA	Comm.	+	-	+	+	-	-	+	+/+	+	-	TRC105	1	2015	20
VEGF-A	LBA	LDA	-	-	-	-	-	-	-	-/-	-	-	Ramucirumab	1	2015	50
virus infectivity	Microscopy	LDA	-	-	-	-	-	-	-	-/-	+	-	Enadenotucirev	1/2a	2019	28

Qualitative validation scores for PD assays from 78 phase 1-3 clinical cancer studies. (+) validation in accordance with the Food and Drug A-dministration bioanalytical method validation guidelines from 2018 (5); (-) not validated; Ref.# = article reference number; validation parameters: I = specificity, II = matrix effects, III = accuracy, IV = dilution linearity, V = dilution integrity, VI = sensitivity/limit of detection, VII = lower limit of quantification, VIII = within- and between day precision, IX = quality controls, X = sample storage stability; ADA = anti-drug antibodies; Ang-2 = Angiopoietin-2; BTK = Bruton’s Tyrosine Kinase; ccK18 = cleaved cytokeratin 18; CD = cluster of differentiation; CgA = chromogranin A; CRP = C-reactive protein; mCTC = mesenchymal circulating tumor cells; CEC = circulating endothelial cells; CTX-1 = C-terminal telopeptide of type I collagen; Comm. = commercial assay; EGF = epithelial growth factor; ELISA = enzyme linked immunosorbent sandwich assay; EPO = Erythropoietin; dFdC-TP = gemcitabine triphosphate; FC = flow cytometry; FGFR = fibroblast growth factor receptor; G-CSF = granulocyte colony stimulating factor; GSH-GSSG = glutathione reduced and oxidized; γ-H2AX = phosphorylated variant histone 2A; HPLC = high performance liquid chromatography; HSP70 = heat shock protein 70; γ-IF = gamma interferon; IGF1R = insulin growth factor receptor; Il = interleukin; LFA = Prolactin Receptor Antagonist antibody; LBA = ligand binding assay; LDA = lab developed assay; LC-MS/MS = liquid chromatography tandem mass spectrometry; MRM-MS = multiple reaction monitoring mass spectrometry; n.d. = not disclosed p-AKT = phosphorylated serine/threonine-protein kinase; PAR = poly-adenosine diphosphate ribose; p-BAD = phosphorylated B-cell lymphoma antagonist of cell death; p-CREB = cAMP Response Element Binding protein; PD1 = programmed death; PDGF = platelet derived growth factor; PD-L1 = programmed death ligand; p-ERK = extracellular-signal-regulated kinase; p-FAK = Focal adhesion kinase; PI3K = phosphatidylinositol 3‑kinase; p-RAD50 = double strand break repair protein; PRAS40 = proline-rich serine/threonine-protein kinase substrate; p-SCR = serum creatinine; RON8 = Macrophage-stimulating 1-receptor; VEGFR = vascular endothelial growth factor receptor; TSA = thymidylate synthase activity; RIA = radioimmunoassay; xMAP = multi-analyte profiling.