Biomarkers for pancreatic cancer: Recent achievements in proteomics and genomics through classical and multivariate statistical methods

Table 4 Genomic based studies

Ref.	Type of marker	Markers	Sample	Study group	Analytical methods	Statistical methods	Performance
68	P	K-ras mutation status and subtypes	endoscopic ultrasound-guided fine-needle aspiration specimens	242 patients	RT-PCR	Kaplan Meier method and Cox proportional hazards regression	Multivariate analysis: CA19-9 C 1000 U/mL (HR = 1.78, 95 %CI: 1.28-2.46, P < 0.01), metastatic stage (HR 2.26, 95 % CI 1.58-3.24, P < 0.01) and mutant-K-ras (HR 1.76, 95 %CI: 1.03-3.01, P = 0.04) negative prognostic factors. Among patients with K-ras mutation subtypes: CA19-9 C 1000 U/mL (HR 1.65, 95%CI: 1.12-2.37, P < 0.01), metastatic stage (HR 2.12, 95%CI: 1.44-3.14, P < 0.01), and G12D or G12R mutations (HR = 1.60, 95%CI: 1.11-2.28) negative prognostic factors for OS
69	P	MicroRNA-21	T	82 resected Korean PDAC cases. Subgroup of patients treated with adjuvant therapy (n = 52)	Protein expression by immunohistochemistry microRNA expression by qRT-PCR	Cox proportional hazards model	Subgroup with adjuvant therapy: lower than median miR-21 expression associated with lower HR for death (HR = 0.316, 95%CI = 0.166-0.600, P = 0.0004) and recurrence (HR = 0.521, 95%CI = 0.280-0.967, P = 0.04). MiR-21: single most predictive biomarker for treatment outcome. No significant association in patients not treated with adjuvant therapy. Independent validation cohort of 45 frozen PDAC tissues from Italian cases treated with adjuvant therapy: lower than median miR-21 expression confirmed to be correlated with longer OS and DFS
71	P	13 putative PDA biomarkers from the public biomarker repository		A survival tissue microarray was constructed comprised of short-term (cancer-specific death ,12 mo, n = 58) and long-term survivors (30 mo, n = 79) who underwent resection for PDA (total, n = 137)	Immunohistochemical analyses; survival tissue microarray (s-TMA)	Wilcoxon rank sum test	Multivariate model: MUC1 (OR = 28.95, 3+ vs negative expression, P = 0.004) and MSLN (OR = 12.47, 3+ vs negative expression, P = 0.01) highly predictive of early cancer-specific death. Pathological factors (size, lymph node metastases, resection margin status, and grade): ORs < 3 and none reached statistical significance. ROC curves used to compare the 4 pathological prognostic features (ROC area = 0.70) to 3 univariate molecular predictors (MUC1, MSLN, MUC2) of survival group (ROC area = 0.80, P = 0.07)
1	P	MTA2 mRNA and protein expression	T	123 PDAC samples and 40 control tissues	qRT-PCR and immunohistochemistry	Kaplan-Meyer curves and Cox analysis	MTA2 mRNA and protein expression levels up-regulated in PC. MTA2 correlated with poor tumor differentiation, TNM stage and lymph node metastasis. Patients with high expression levels of MTA2 showed lower OS. MTA2: independent prognostic factor.
72	D	Leukocyte DNA Methylation	Blood	Phase I: 132 never-smoker PaC patients and 60 never-smoker healthy controls. Phase II: validation of 88 of 96 phase I selected CpG sites in 240 PaC cases and 240 matched controls	DNA array	Wilcoxon Rank Sum tests and likelihood penalized logistic regression models	Significant differences found in 110 CpG sites (FDR < 0.05). Phase II: 88 of 96 phase I selected CpG sites validated in 240 PaC cases and 240 matched controls (P < 0.05). Prediction model: 5 CpG sites (IL10_P348, LCN2_P86, ZAP70_P220, AIM2_P624, TAL1_P817) discriminated PaC from controls (C = 0.85 in phase I; 0.76 in phase II). One CpG site (LCN2_P86) could discriminate resectable patients from controls (C = 0.78 in phase I; 0.74 in phase II). 3 CpG sites identified (AGXT_P180_F, ALOX12_E85_R, JAK3_P1075_R) where the methylation levels were significantly associated with SNPs (FDR < 0.05)
73	D	cell-surface targets	T	28 PC specimens and 4 normal pancreas tissue samples. Expression in normal tissues evaluated by array data from 103 samples representing 28 organ sites as well as mining published data	Complementary assays of mRNA expression. Immunohistochemistry. qRT-PCR	-	170 unique targets highly expressed in 2 or more PC specimens and not expressed in normal pancreas samples. Two targets (TLR2 and ABCC3) further validated for protein expression by tissue microarray based immunohistochemistry have potential for the development of diagnostic imaging and therapeutic agents for PC
74	D	Differentially expressed genes	Blood	25 patients diagnosed with PC and diabetes, 27 patients with PC without diabetes, 25 patients with diabetes mellitus > 5 yr, and 25 healthy controls. Results further validated for 101 blood samples	Microarray and qRT-PCR	-	58 genes found to be unique in patients with cancer-associated diabetes, including 23 up-regulated and 35 down-regulated genes. 11 up-regulated genes further validated by RT-PCR; 2 of these (VNN1 and MMP9) selected for logistic regression analysis. The combination of VNN1 and MMP9 showed the best discrimination of cancer-associated diabetes from type 2 diabetes. The protein expression of MMP9 and VNN1 was in accordance with the gene expression

Type of marker: P: Prognostic/predictive; D: Diagnostic; Sample: S: Serum; P: Plasma; T: Tissue; PDAC: Pancreatic ductal adenocarcinoma; VNN1: Vanin-1; MMP9: Matrix metalloproteinase 9.