Gallbladder Cancer: Approaches to Biomarker Discovery

Tekcham DS and Tiwari PK*

Centre for Genomics, Molecular and Human Genetics, Jiwaji University, Gwalior, India

*Corresponding author: Tiwari PK, Centre for Genomics, Molecular and Human Genetics, Jiwaji University, Gwalior-474 011, Madhya Pradesh, India

Received: September 04, 2015; Accepted: October 29, 2015; Published: November 05, 2015

Abstract

Gallbladder cancer is a relatively uncommon global health issue, affecting most commonly the middle-aged women. Due to lack of early diagnostic marker and late detection of tumor, survival of GBC patients is compromised. Surgical resection of gallbladder is the only option for treatment. Here, we present the updates on molecular mechanism of GBC, current technological strategies, limitations and future prospects of biomarker discovery. The association of genetic, cytogenetic and epigenetic mechanisms with GBC has brought tremendous alteration in the expression of various protein coding genes. The recent advancements in high throughput technologies have replaced the conventional methods and now being considered as major tools for biomarker discovery. The earlier efforts on the identification of chromosomal loci harboring loss of heterozygosity alleles to use of both conventional and advanced technologies all, have identified some 4000 plus molecules from various biological sources of GBC (tissue, blood and cell lines). In this review, we have updated the list of molecules identified by major high throughput as well as conventional methods, which are currently the main targets in the discovery of diagnostic and or therapeutic biomarkers for GBC.

Keywords: Gallbladder; Cancer; Biomarker

Introduction

Gallbladder cancer is an uncommon cancer and is a female biased health issue, affecting the middle age group more often. In 2015, 10910 new GBC cases and 3700 estimated deaths were predicted in the United States [1]. The highest incidence of GBC is reported from Delhi, India (21.5/1,00,000), followed by South Karachi, Pakistan (13.8/1,00,000) and Quito, Ecuador (12.9/1,00,000) [2]. Our epidemiological study in north central Indian region showed significantly high incidence of GBC (7.8/1,00,000) [3]. The Indian Council of Medical Research (ICMR) Population Based Cancer Registry (ICMR-PBCR 2003-04) [4] reported the highest incidence of GBC in female to be 10.2 per lakh population, in Kamrup, Assam, India. Variations in the geographical distribution of the incidence of GBC reflect distinct ethnic (genetic) association. Gallstone disease is a potential risk factor of GBC. Increase in the number and size of the stones is directly related to the risk of developing GBC [5]. Reports have shown that 60% [6] to more than 80% GBC cases possess stone (multiple/single) [7]. Hundreds of genes have been identified to be significantly associated with GBC and having potential to be developed as useful diagnostic/ prognostic and even therapeutic biomarkers. Discovery of useful non-invasive biomarkers have always been desirable for diagnostic purposes, which are supposed to be safe and rapid [8]. Although our effort to dissect out the mechanism of gallbladder tumorigenesis is in progress, there are many challenges at various fronts. Hence, detailed investigations are needed in well classified group of samples, employing advance high through put technologies to understand the role of those genes, showing association with the GBC pathogenesis. This review is an attempt to summarize our current understanding on gallbladder tumorigenesis, use of technological strategies in search of clinically relevant biomarkers for early diagnosis, limitations and future prospects.

Mechanisms of gallbladder tumorigenesis

Various reports claimed Gall Stone Diseases (GSD) to be genetically modulated [9]. Upcoming information on family based investigations has replaced the quote as “GSD to be genetically associated” [10-11]. Formation of gallstone and Anomalous Pancreato-Biliary Duct Junction (APBDJ) are two pathological states for gallbladder tumor formation [12-14]. As GSD is an early pathological event, probably towards GBC, it is interesting to reveal its possible genetic association. We can call GSD as driver of GBC. Here, our effort is to present an update on the mechanisms that lead to GBC.

Genetic basis: Genetic association of GBC has become clear from various reports. Initially, the use of PCR based Loss of Heterozygosity (LOH) method could identify the loci, which harbor susceptibility genetic markers. Some of the earlier studies using PCR based allelotyping method detected more than 10 LOH-loci in GBC from Chile [15-16]. Restriction Fragment Length Polymorphism-PCR (RFLP-PCR) or gene sequencing is one of the most conventional methods used for identification of genetic variants [17-18]. Genomewide allelotyping identified 21 hot spot loci, including RNF4, SH3BP2, AF6q21, CD24, p73, DUTT1, FHIT, RAR-β, BLIMP1, CCNC, SMOH, PDGFR b-like, N33, FEZ1, p16Ink4/CDKN2, p15INK4b/CDKN2, NBCCS, DEC1, TSC1, CACNB2, MEN-1, TP53, STK11/LKB1 and NF2, distributed on 16 different chromosomes in GBC [19]. In a genome wide study in Japanese population, a genetic variant of Deleted in Colon Cancer (DCC) was found linked with Gallbladder Cancer [20]. Earlier studies claimed mutations to be responsible for their loss of expression of K-RAS [21], TP53 [22] and CDKN2A [15] in GBC, although later other mechanisms were also suggested. Hundreds of genes are expressed or regulated as genetic variants in GBC. Srivastava et al., (2011), in a meta-analysis, showed possible risk and association of various genetic variants with GBC [18]. Significant association of Prostate Stem Cell Antigen (PSCA) gene variants with GBC is reported in female patients in North Indian population [23]. In the same population group, increased risk of GBC was also found associated where genetic variants of Matrix Metalloproteinase (MMP- 2, 7, 9), tissue inhibitor of metalloproteinase (TIMP-2) [24], CYP1A1 and CYP1B1 [25] were pre-dominant. The ApoB-100 X+X+ genotype is also suggested to be associated with reduced risk of GBC [26]. Micro Satellite Instability (MSI), a common genetic mechanism of change in tandem repeats, is commonly observed in several genes, like TGFβR-II [27], BAT25, BAT26, D2S123 and D17S250 [28] in GBC. Loss of heterozygosity is one such mechanism suggested to play a key role in GBC [15,19,29] (Table 1).

Table 1: Selected list of LOH/genes/SNPs identified by high throughput technologies for biomarker identification in GBC and GSD.




  
    SL.    No. 
    No.    of Molecules Identified 
    Significant    markers 
    Biopsy 
    Technology 
    Status 
    References 
  
  
                  LOH/Genes/SNP 
                 Genomics 
  
  
    1 
    ------- 
    KRAS,    TP53, ERBB3, EGFR, ERBB2, ERBB4 
    Tissue 
    WES    & UDS 
    GBC 
    [54] 
  
  
    2 
    14 
    IDH1,    KRAS, NRAS, PIK3CA, MET 
    Tissue 
    SMA 
    GBC 
    [57] 
  
  
    3 
    26 
    TP53,    STK11, RICTOR, TSC2 
    Tissue 
    NGS 
    GBC 
    [57] 
  
  
    4 
    ------ 
    D1S1597    and D1S407 
    Tissue 
    GWAS 
    GSD 
    [58] 
  
  
    5 
    130 
    DCC 
    Tissue 
    GWAS 
    GBC 
    [20] 
  
  
    6 
    21 hot    spots 
    p73,    DUTT1; FHIT, RARβ, RNF4; SH3BP2, AF6q21, CD24, BLIMP1, CCNC, TSC1, SMOH,    PDGFR b-like, N33, p16, p15, NBCCS, DEC1, CACNB2, MEN-1, TP53, STK11/LKB1,    NF2, FEZ1 
    Tissue 
    Genome-wide    allelotyping 
    GBC 
    [19] 
  
  
    7 
    1281 
    RRM2,    PTTG1, TYMS, CDC2, CCNB2, RACGAP1, SHMT2, ANK2, ACACB, MYOM1, ITGA7, CDKN1C    ALDH1A2, CNN1, CES1, DES 
    Tissue 
    array    CGH & RTPCR 
    GBC 
    [59] 
  
  
    8 
    5 
    APC,    CDKN2A, ESR1, PGP9.5 and SSBP2 
    Tissue 
    ELISA    based & qMSPCR GBC & GSD 
    GBC    & GSD 
    [60]



Table 1: Selected list of LOH/genes/SNPs identified by high throughput technologies for biomarker identification in GBC and GSD.

Epigenetic basis: Introduction of methylation specific PCR to cancer research in 1996 opened the new area of “epigenetics” in cancer [30]. The large number of reports published so far has demonstrated epigenetics to be equally important as genetic mechanisms in the process of tumorigenesis. DNA methylation, histone modification (acetylation, phosphorylation, ubiquitination, sumoylation, methylation, etc.,), RNAi, etc., are a few examples of the epigenetic mechanisms. Much less is known about the role of epigenetics in GBC. Promoter hypermethylation of p16, p73, APC, MGMT, hMLH, RAR [31], SHP1, 3-OST-2, CDH13, p15, CDH1, RUNX3, RIZ1, p16INK4A and HPP1 genes [32] are commonly reported in GBC. We also screened the methylation pattern of several target tumor associated genes in the GBC patients of north central India, which showed APC [33] and PTEN (manuscript submitted) to be epigenetically down regulated in GBC. Among other genes analyzed, Maspin and 14-3-3 sigma genes also showed promoter methylation in most of the GBC cases [34]. Recently, Feng et al. demonstrated regulation of proliferation of GBC cells by histone acetyl transferase enzyme genes, ATF2, KAT5 and EPC1 [35]. A list of methylated genes in GBC may be found in Letelier et al [36]. The role of histone modification in GBC is still not fully known. Recent reports have implicated the role of small non-coding miRNAs, like miR-215-8p [37], miR-130a [38], miR-138 [39], miR-146b-5p [40], miR-26a [41], miR-1, miR-145 [42] and miR-20a [43] in GBC. More extensive investigation is required to understand fully the role of epigenetics in GBC (Table 2).

Sl. No.	No. of Molecules identified	Significant markers	Biopsy		Technology	Status	References
*MicroRNAs*				*Transcriptomics*
1	---	miR-26, miR-34a	Tissue/cell line		Validation Assays	GBC	[41,62]
2	---	miR-146b-5p	Tissue/cell lines		Validation Assay	GBC	[40]
3	11	let-7a, miR-21, miR-187, miR-143, miR- 202, and miR-335	Blood Serum		Micro Array	GBC	[56]
4	17	miR-210	Tissue		Illumina Sequencing & RTPCR	GBC	[63]
5	17/880	miR-20a	Tissue		miRNA Library Screening by High Content Screening	GBC	[64]
6	36/481	miR-1, miR-133, miR-143, miR-145	Tissue		Microarray & RTPCR	GBC	[42]
7	---	miR-21, miR-142-3p, miR-122, miR- 142-5p,miR-223	Mice Tissue		Microarray & RTPCR	GBC	[65]

Sl. No.	No .of Molecules identified	Significant markers	Biopsy		Technology	Status	References
*Proteins*				*Proteomic*
1	2550	MARCKS, BSG, EPCAM, ICAM1, AXL, PAK1, ERBB2, NDRG1, CIAPIN1, ALCAM	Human bile		LC-MS/MS	Cholecystitis	[44]
2	544	S100A8	Tissue		2D & MS	GBC	[66]
3	495	S100A8	Tissue		2D & MS	Cholecystitis	[66]
4	24	S100A10, Haptoglobin, Cystatin-B, Profilin-1 and superoxide dismutase	Serum		MALDI-TOF-MS	GBC	[46]
5	46	Annexin A3, PEBP1	Tissue		2D & MS	GBC	[45,67]
6	26	CLIC1, Ezrin, Vimentin, Annexin A3, WD repeat domain 1, Triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, Heterogeneous nuclear ribonucleoprotein K, Glutamate dehydrogenase 1, Proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta	Cell line		2D & MS	GBC	[57]
7	6	ANXA4, HSP90 beta, Dync1h1, ACTA2, Serum albumin	Tissue		MALDI-TOF-MS	GBC	[68]
8	2575	NUCKS1, DMBT1, Mucin 5A, MUC13, LAMB3, PSAP, HMGB2, NEFH, S100AS, TAGL	Tissue		LC-MS/MS	GBC	[51]
9	26	Vimentin	Cell line/in vivo		2D & MS	GBC	[69]

Gallbladder Cancer: Approaches to Biomarker Discovery

Abstract

Introduction

Mechanisms of gallbladder tumorigenesis

Alterations in Gene Expression in Gallbladder Cancer

Technological Strategies in Biomarker Identification

Limitations of strategies

Future perspectives

References