Table 4.

Biomarkers reported more than once for the same tumour type and panels adopting a single-gate (reversed-flow) design

References	Recruitment setting	Cases	Controls	Outcomes (Sensitivity, specificity, AUC where available)
1. Measures of diagnostic performance available for individual biomarkers, in studies adopting a single-gate design
Apolipoprotein AII-AT/ATQ alone and in combination with CA19-9 (pancreatic cancer)
Honda et al. [106]	EPIC cohort (population-based study)	156 PaC Median age 58.1 (34.9–75.7) 53% male Staging: 13 localised, 73 metastatic, 69 NA	213 HC Median age 58.0 (34.5–75.4) 53% male (matched to cases)	Measures for months prior to diagnosis (lag times): up to 6 months, > 6–18, 18, > 18–36 and > 36–40 months For ApoAII-AT/ATQ alone, 2 cut-off points Sensitivity, range 0.04–0.25 AUC, range 0.52–0.62 For ApoAII-AT/ATQ plus CA19-9, 2 cut-off points Sensitivity, range 0.07–0.57 Specificity, range 0.96–0.98 AUC, range 0.56–0.78
Honda et al. [105]	Cohort 1: National Cancer Centre Hospital	131 IDACP Mean age 68.8 (9.01) 55% male Staging: most at advanced stages	131 HC Mean age 62.5 (10.8) 52% male	Measures for ELISA and mass spectrometric analysis, also according to tumour staging For ApoAII-ATQ/AT alone, 1 cut-off point AUC, range 0.856–0.946 For ApoAII-AT/ATQ plus CA19-9, 1 cut-off point each Sensitivity, 95.4% (cohort 2) Specificity, 98.3% (cohort 2)
	Cohort 2: Seven Medical Institutions	155 IDACP Age and sex NA Staging: majority advanced stages	57 pancreatic disease other than IDACP Age and sex NA
	Cohort 3: NCI-EDRN pancreatic reference set	98 PaC Age and sex NA Staging: all early stages	62 CP, 31 acute benign biliary obstruction, 61 HC Age and sex NA
Honda et al. [104]	Cohort 1: National Cancer Hospital and Medical University Hospital	Does not meet criteria as used to calculate first measures of performance	Does not meet criteria as used to calculate first measures of performance	Measures provided according to tumour staging For ApoAII-AT/ATQ alone, 1 cut-off point AUC, 0.953 (cohort 3) For ApoAII-AT/ATQ plus CIII-0, and CA19-9, 1 cut-off point (cohort 4) Sensitivity, range 91.60–94.20% Specificity, 93.22% (same for all)
	Cohort 2: National Cancer Hospital	Does not meet criteria as there were only 41 controls	Does not meet criteria as there were only 41 controls
	Cohort 3: Department of General Surgery	52 PaC Mean age 63.1 (9.85) 56% male Staging NA	53 HC and 58 CP HC mean age 39.1 (15.6), CP 50.3 (8.9) HC 59% male, CP 74% male
	Cohort 4: Seven Medical Institutions	249 PDAC and 18 other malignant tumour of the pancreas PDAC mean age 64.4 (9.1), other 68.3 (9.7) PDAC 59% male, other 67% male Staging NA	128 HC, 38 benign tumour/cyst and 14 CP HC mean 46.6 (16.8), benign tumour/cyst 63.5 (11.0), CP 60.2 (10.2) HC 65% male, benign tumour/cyst 45% male, CP 86% male
Pepsinogen (PGI and PGI/II ratio) (gastric cancer)
Gantuya et al. [29]	National Cancer Centre Hospital	50 GC (54% w/ H. pylori) No information on age and sex Staging NA	752 non-cancer (302 antrum limited CG and/or atrophy and 450 corpus CG and/or atrophy (77% w/ H. pylori Mean age: 53.8 (SD 1, 27–78) 31% male	For PGI, optimal cut-off point Sensitivity, 70% Specificity, 70% AUC, 0.76 For PGI/II ratio, optimal cut-off point Sensitivity, 66% Specificity, 65% AUC, 0.70
Kang et al. [40]	National University Hospital	380 GC (intestinal and diffuse type) Age and sex not available for cases only No information on staging	172 BGU, 119 DU, 107 dysplasia Age and sex not available for controls only	Measures according to tumour type only (intestinal or diffuse) For PGI, 1 cut-off point Sensitivity, 77.7% (intestinal), 64.7% (diffuse) Specificity, 20.2% (intestinal), 20.2% (diffuse) For PGI/II ratio, 1 cut-off point Sensitivity, 62.3% (intestinal), 55.8% (diffuse) Specificity, 61.0% (intestinal), 61.0% (diffuse)
Kikuchi et al. [41]	University Outpatient Clinic	122 GC Age: 68.2 years (9.7) 74% male Staging NA	16 GU or DU, 17 superficial gastritis, 66 CAG, 79 no abnormality Age: 56.2 years (14.9) 55% male	Measures combining normal and non-malignant conditions Negative or positive PG test For PGI and PGI/II ratio, strict or conventional cut-off point Sensitivity, 41.3% (strict), 77.9% (conventional) Specificity, 90.4% (strict), 61.8% (conventional)
Yanaoka et al. [76]	Employees in annual health screening programme	63 GC Age: 50.3–51.8 (mean range) 100% male 86% early, 14% late stages	5146 HC Mean age: 49.2 (4.7) 100% male	or PGI and PGI/II ratio, 3 cut-off points Sensitivity, range 27.0–58.7% Specificity, range 73.4–92.0%
2. Measures of diagnostic performance available for established biomarkers combined with novel biomarkers not shown above, in studies adopting a single-gate design
CA19-9 (pancreatic cancer)
O’Brien et al. [121]	UKCTOCS screening cohort	101 PaC Age NA for validation 100% female Staging NA	184 HC Age N/A for validation 100% female	Measures according to time to diagnosis: 0–4 years, 0–2 years; 1–4 years For CA19-9 (4 cut-off points) plus CA125 (3 cut-off points) Sensitivity, range 23.1–53.1% Specificity, range 71.6–92.6%
Tavano et al. [132]	Hospital (Gastroenterology, Surgery & Oncology)	74 PaC Median age 69 (61–76) 54% male Staging NA for validation	117 HC Median age 62 (55–70) 45% male	For CA19-9 plus miR-1290, 1 cut-off point (each) Sensitivity, 83.8% Specificity, 96.6% AUC, 0.923
Zhou et al. [138]	Gastroenterology Department in Hospital	152 PaC Mean age 56 (SD 13.5) 67% male Staging: 5 IA, 12 IB, 36 IIA, 20 IIB, 40 III, 39 IV	96 HC, 91 CP, 20 pre-malignancies Mean age: HC 58 (7.6), CP 58 (15.0), pre-malignancies 60 (11.3) HC 75% male; CP 57% male; pre-malignancy 75% male	For CA19-9 plus MIC-1 and ULBP2, 1 cut-off point (each) AUC 0.982 (PaC and CP only) For CA19-9 plus MIC-1, 1 cut-off point (each) AUC 0.932 (PaC and CP only) For CA19-9 plus ULBP2, 1 cut-off point (each) AUC 0.953 (PaC and CP only)
3. Measures of diagnostic performance available for a panel only in studies adopting a single-gate design (all reversed-flow)
Different panels (pancreatic cancer)a
Balasenthil et al. [89]	NCI-EDRN pancreatic reference set	98 PaC (52 w/o diabetes or pancreatitis) Age and sex not available Staging: 7 IA, 8 IB, 1 II, 40 IIA and 42 IIB	62 CP, 31 acute biliary obstruction, 61 HC (50 w/o diabetes or pancreatitis) Age and sex not available	Measures for PaC vs. HC, PaC vs. CP, PaC w/o diabetes or pancreatitis vs. HC w/o diabetes or pancreatitis, and according to staging For CA19-9 plus TFPI and TNC-FN III-C, 2 cut-off points Sensitivity, range 0.73–0.81 Specificity, range 0.71–0.84 AUC, range 0.75–0.89
Mellby et al. [119]	Patients referred to Medical Centre for symptomatic pancreatic disease	2 cohorts; one for validation (US cohort) 143 PaC patients Median age only by staging; range 24–87 57% male Staging: 15 I, 75 II, 15 III and 38 IV	219 HC, 57 CP HC median age 63.0 (24–86), CP 55.5 (32–81) HC 53% male, CP 46% male	Measures available for stages I + II combined For 29-panel signature (no established biomarkers): Sensitivity, 95% Specificity, 93% AUC, 0.963 (PaC vs. HC) and 0.840 (Pac vs. CP)

ACG atrophic chronic gastritis, ApoAII-AT/ATQ apolipoprotein AII-AT/ATQ, apoCIII-0 apolipoprotein CIII-0, BGU benign gastric ulcer, DU duodenal ulcer, CG chronic gastritis, CP chronic pancreatitis, EPIC European Prospective Investigation into Cancer and Nutrition, GC gastric cancer, GU gastric ulcer, IDACP invasive ductal adenocarcinoma of pancreas, MIC macrophage-inhibitory cytokine 1, MPV mean platelet volume, NA not available, NCI-EDRN National Cancer Institute Early Detection Research Network, PaC pancreatic cancer, PDAC pancreatic ductal adenocarcinoma, PDW platelet distribution width, PGI/II serum pepsinogen I/II, PPV positive predictive value, TFPI plasma tissue factor pathway inhibitor, NTC-FN III-C tenascin-C, UKCTOCS UK Collaborative Trial of Ovarian Cancer Screening, ULBP2 UL16 binding protein 2

^aLeelawat et al. [166] also adopted a reversed-flow design but was not added as it was the only study investigating CA19-9 for cholangiocarcinoma