Early detection of gallbladder cancer: Current status and future perspectives

Table 1 Patients at high risk of developing gallbladder cancer

Serial number	High risk factors	Ref.
1	Age (≥ 50 years), socioeconomic status (≤ below poverty line), bowel habits (≤ once a day), tap water drinking, number of pregnancies (≥ 3 pregnancies), multiparity (≥ 3 babies)	[7]
2	Female sex	[8]
3	Residence in the Gangetic belt, consumption of tea, tobacco, joint family structure, chemical exposure, fried food, high levels of secondary bile salts	[9]
4	Females with more than two children, mustard oil consumption, low socioeconomic strata age of menarche less than 14 years, age of the first child birth less than 20 years, Presence of gall stone	[10]
5	Long-standing gallstone disease, female	[11]
6	Female, older age, solitary stones and stones size more than one centimetre	[12]
7	Increase in the number and size of the stones	[13]
8	Women, long history of gall stone disease	[14]
9	Weight, volume and size of the stones increases the changes in the gall bladder mucosa changes from cholecystitis, hyperplasia, metaplasia, dysplasia, to carcinoma	[15]
10	Stone size, solitary polyps with a size of greater than 1 cm that are echogenic, sessile, and high cell density, AJPBD, porcelain gallbladder	[16]
11	Patients with history of salmonella and helicobacter infection	[17]
12	Primary sclerosing cholangitis	[18]
13	Cholelithiasis, females with gallstones in their sixth decade, multiple stones	[19]
14	Higher stone volume	[20]
15	High soil arsenic levels, residence in gangetic belt	[21]
16	Smoking, cholelithiasis, alcohol consumption, typhoid in the past, post-menopausal women	[22]
17	Helicobacter pylori infection	[23]
18	Females, consumption of mustard oil, family history, low socioeconomic status and drinking water from hand pump	[24]

AJPBD: Anomalous junction of pancreaticobiliary ducts.

Table 2 Summary of various imaging modalities for identifying malignant gallbladder pathology

Imaging modalities	Features suggestive of malignancy	References
USG	Localized thickening, mass and a stone GB lumen with localized thickening	[50]
	Mass along with thickening	[51]
	Polypoidal mass with wall thickening > 1 cm, hypoechogenicity, internal hypoechoic foci	[52]
	Loss of layered structure and enhancement of wall	[53]
	Solitary gallstone, displaced stone, intraluminal mass, GB-replacing or invasive mass, discontinuity of the mucosal echo	[54]
CEUS	Arterial phase inhomogeneous hyperenhancement, venous phase hypoenhancement and disruption of GB wall layer structure	[55]
	Rapid GB wall blood flow and the irregularity of color signal patterns on doppler imaging, and heterogeneous enhancement in the venous phase, focal thickening, discontinuity and irregularity of the innermost hyperechoic layer and irregular or disrupted GB wall layer structure	[56]
	Differences in enhancement direction, vascular morphology, serous layer continuity, wash-out time and mural layering in the venous phase	[57]
	An irregular shape, branched intralesional vessels, and hypo-enhancement in the late phase	[58]
	Homogeneous enhancement in the arterial phase, followed by interrupted inner layer, early washout (≤ 40 seconds), and wall thickness > 1.6 cm	[59]
CECT	The thicknesses of the inner and outer layers (“thick” enhancing inner layer > or = 2.6 mm, “thin” outer layer < or = 3.4 mm), strong enhancement of the inner wall, Irregular contour GB wall, layering pattern - two-layer pattern with a strongly enhancing thick inner layer and weakly enhancing or nonenhancing outer layer and the one-layer pattern with a heterogeneously enhancing thick layer	[60]
	Wall irregularity, focal wall thickening, discontinuous mucosa, submucosal edema, polypoid mass, direct invasion to adjacent organ, biliary obstruction, regional and paraaortic lymphadenopathy and distant metastasis	[61]
	The thickened GB wall with one-layer heterogeneous enhancement (type 1)
	Mass replacing GB, diffuse/focal GB wall thickening and polypoidal mass, associated cholelithiasis, liver infiltration, intra hepatic biliary dilatation, liver metastases, portal vein invasion, antroduodenal and hepatic flexure involvement	[62]
	Heterogeneous peripheral and central enhancement in arterial phase	[63]
MRI	Heterogeneous enhancement, indistinct interface with the liver, and diffusion restriction
	T2 moderate hyperintensity of the thickened wall, papillary appearance, and diffusion restriction	[44]
	Discontinuous enhancing mucosal line, earlier enhancement of wall, diffusion restriction, lower mean ADC in malignant wall	[64]
	Diffusion restriction	[65]
	Low ADC and diffusion restriction	[66]
	Diffusion-weighted examination with a high b value	[67]
PET Scan	Higher SUV uptake	[68]
	Delayed uptake in dual phase PET

ADC: Apparent diffusion coefficient; MRI: Magnetic resonance imaging; CECT: Contrast-enhanced computed tomography; USG: Ultrasonography; CEUS: Contrast-enhanced ultrasound; PET: Positron emission tomography; SUV: Standardized uptake value; GB: Gallbladder.

Table 3 Sensitivity, specificity and limitation of traditional imaging modalities for early detection of gall bladder cancer

Imaging modalities	SN	SP	Accuracy	Advantages	Limitations	References
USG	65%-94%	70%-95%	80%-90%	Inexpensive, non-invasive, portable, and widely available	Low accuracy in differentiating benign vs malignant GB wall thickening	[45,69,70]
				Can flag suspicious case	Malignant small sessile polyps
					Operator dependent
					Nodal involvement
CEUS	90%-100%	90%-95%	55%-90%	Portable	Smaller lesion gives false positive results so less sensitive for smaller lesions	[55,57,71-74]
				Better modality for detecting vascularity and lesion characterization	Operator dependent
					Artefacts
					Not available widely
CECT	70%-100%	40%-100%	75%-95%	Better characterization staging	Limited sensitivity in small polyps, T1 lesions, thick walled	[60,61,75-77]
				Better anatomical detail	High cost
				Lymph node involvement	Radiation exposure
					Poor specificity to differentiate XGC, AC and GBC
					Not available in rural areas
MRI	70%-88%	60%-70%	92%	Excellent soft tissue contrast, good for delineating bile duct involvement	Miss early lesions with subtle findings	[78-80]
					Overdiagnosis of benign lesions
					High cost
					Not readily available in rural areas
PET	70%-80%	80%-85%	50%-70%	Only for distant metastasis	Cannot accurately differentiate benign inflammation and malignant thickening	[81,82]
					High cost and not readily available

SN: Sensitivity; SP: Specificity; MRI: Magnetic resonance imaging; CECT: Contrast-enhanced computed tomography; USG: Ultrasonography; CEUS: Contrast-enhanced ultrasound; PET: Positron emission tomography; SUV: Standardized uptake value; GB: Gall bladder; GBC: Gall bladder cancer; AC: Acute cholecystitis; XGC: Xantho granulomatous cholecystitis.

Table 4 Imaging findings of early gall bladder cancer on various modalities

Modalities	Imaging characteristics
USG	Polyp
	Focal thickening
	Asymmetric thickening
	Subtle GB wall thickening
	Irregular thickening
CECT	Subtle wall thickening
	Localized thickening > 4 mm
	Small polyp
CEUS	Dotted intralesional blood vessels
	Arterial phase inhomogeneous hyperenhancement, venous phase hypoenhancement and disruption of GB wall layer structure
RTE	Higher mean shear wave elasticity
PET scan	Higher SUV uptake

USG: Ultrasonography; CECT: Contrast-enhanced computed tomography; CEUS: Contrast-enhanced ultrasound; PET: Positron emission tomography; SUV: Standardized uptake value; GB: Gall bladder; RTE: Real-time elastography.

Table 5 Malignant characteristics of gall bladder polyp(s)

Modalities	Factors favoring malignant polyps	References
Clinical	Endemic areas, associated with PSC, old age > 60 years	[90-94]
USG	Single lobular surface, vascular core, hypo-echoic polyp and hypoechoic foci or a polyp size of greater than 1 cm, associated gall stones, sessile polyp, localized GB wall thickening, associated gall stone disease, focal gallbladder wall thickening > 4 mm, growth 2-4 mm/year	[92,95-99]
CEUS	Fast-in and “fast-out” enhancement pattern, hyper-enhancement in comparison to the GB wall in the arterial phase, wash-out time ≤ 40 seconds, GB wall destruction, and hepatic parenchymal infiltration, diffuse and branched types of contrast enhancement	[74,100]
CECT	Enhancement of polyp, mass filling GB lumen, liver infiltration, surrounding lymphadenopathy, associated asymmetric thickening,	[52]
PET scan	High SUV max	[89]

CECT: Contrast-enhanced computed tomography; USG: Ultrasonography; CEUS: Contrast-enhanced ultrasound; PET: Positron emission tomography; SUV: Standardized uptake value; GB: Gallbladder; RTE: Real-time elastography; PSC: Primary sclerosing cholangitis.

Table 6 Benign vs malignant gall bladder wall thickening

Modalities	Benign wall thickening	Malignant wall thickening
USG	Diffuse and symmetric	Focal and asymmetric
	Intact mucosa	Discontinuous mucosa
	Layered GB wall	Loss of layering
	Low mean flow velocity and peak systolic velocity	High mean flow velocity and Peak systolic velocity
	Low shear wave velocity	High shear wave velocity
	Liver parenchyma infiltration absent	Liver parenchyma infiltration present
CEUS	Homogenous arterial phase enhancement	Non-homogenous arterial phase enhancement
	Tortuous intralesional vascularity	Dotted intralesional vascularity
	Delayed washout	Early washout
CECT	Homogenous enhancement	Heterogenous enhancement
	If layering present inner layer is enhancing	If layering present inner layer enhancing
	Lymphadenopathy usually absent	Present
	Symmetric	Asymmetric
MRI	On T2, thin hypointense inner layer and thick hyperintense outer layer or multiple T2 hyperintense foci in wall	Diffuse nodular thickening without
	Delayed enhancement	Layering
	High ADC	Early enhancement in contrast phase
		Low ADC

MRI: Magnetic resonance imaging; CECT: Contrast-enhanced computed tomography; USG: Ultrasonography; CEUS: Contrast-enhanced ultrasound; GB: Gall bladder; ADC: Apparent diffusion coefficient.

Table 7 Major studies on association of various serological markers in gall bladder cancer

Serial number	Biological markers	Cut off value	Sensitivity	Specificity	Ref.
1	CEA	5 ng/mL	52%	55%	[115]
	CA19-9	37 U/mL	74%	82%
	CYFRA 21-1	2.7 ng/mL	76%	79%
	MMP7	7.5 ng/mL	78%	77%
	Combination of all four	-	92%	96%
2	CEA	1.95 ng/mL	90.2%	35.29%	[116]
	CA19-9	26 U/mL	58.82%	83.82%
	Combination of the two	-	90.2%	88.24%
3	CEA	10 μg/L	11.5%	97.4%	[117]
	CA19-9	39 U/mL	71.7%	96.1%
	CA242	15 U/mL	64.1%	98.7%
	CA125	35 U/mL	44.8%	96.2%
4	CA 242	20 U/mL	64%	84%	[118]
	CEA	5 U/mL	61%	44%
	CA 19–9	35 U/mL	17%	67%
5	CYFRA 21-1	3.27 ng/mL	93.7%	96.2%	[119]
	CYFRA 21-1	2.61 ng/mL	74.6%	84.6%
	CYFRA 21-1	3.27 ng/mL	75.6%	96.2%
	CYFRA 21-1	2.27 ng/mL	71.0%	71.2 %
6	CA 19-9	39 U/mL	71.3%	90.0%	[120]
	CA 125	36 U/mL	38.8%	93.3%
	CEA	10.36 U/mL	12.5%	92.5%
	CA 242	15 U/mL	86.3%	90.0%
7	CA 19-9	252.31 U/mL	100%	98.90%	[121]
	CA 125	92.19 U/mL	100%	94.50%
8	CA 19-9	250 U/mL	76.3%	70.8%	[122]

CEA: Carcinoembryonic antigen; CA: Carbohydrate antigen; CYFRA 21-1: Cytokeratin-19 fragment antigen 21-1; MMP7: Matrix metalloproteinase-7.

Table 8 Genetic biomarkers associated with gall bladder cancer

Types of genetic markers	Genes	Ref.
Inflammatory markers	CR1, PTGS2 (cyclooxygenase), TLR, sTNFR2, IL-6, sTNFR1, CCL20, VCAM-1, IL-16, G-CSF, TGFb1, IL-8, MMP-2,7,9	[128-134]
Metabolic pathway genes	CYP1A1, Ile462Val (rs1048943), (Japanese and Hungarian population), IVS1 + 606 (rs2606345) T allele of CYP1A1 (Chinese population), GSTM1 (Bolivian population), MTHFR, APOB, NAT2, GSTT1, GSTP1 CYP17, LDLR, LPL, ALOX5, ABCG8, CETP, LAPAP1, ApoB, CYP17A1ADRB3	[135-146]
DNA repair pathway genes	CC genotype of TP53 (India), ERCC2, IVS1 + 9G > C in the MSH2, Ser326Cys in the 8-OGG1, EX5-25C>T in the O6-aky guanine DNA acyltransferase (MGMT), APEX1, RAD23B, FEN1	[147-150]
Hormone pathway genes	CCKAR, ESR1, ESR2, CYP1A1, CYP19A, HSD3B2, RXR- a PPARD	[151-156]
Apoptosis pathway	DR4 haplotype C rs20575 A rs20576 A rs6557634, caspase-8	[157-158]
Cancer Stem cell gene	CD44, NANOG, ALCAM, EpCAM, SOX-2, OCT-4, and NANOG	[159]
MiRNA	hsa-miR-146a, hsa-mir-196a2, hsa-mir-499 miR-27a (rs895819) A>G	[160,161]
WNT signalling pathway	SFRP4, DKK2, DKK3, APC, AXIN-2, Β-CATENIN, GLI-1	[162]
Genome-wide association study	SERPINB5, BCL10, CD44, ARHGEF11 SERPINB2, RELA, PAK4, PPARD and BUB1B. SNP rs7504990 in the DCC (Japan), SNPs in ABCB1 and ABCB4 genes (India)	[163-165]
Others	KRAS (codon 12,13,61), c-erb-B2, ACE I/D, DNMT3B, VDR	[166-171]

Table 9 Genetic biomarkers that mutated in early carcinogenesis and can be used for early diagnosis

Genes	Mutation	Method of identification	Role in early diagnosis	Ref.
TLR2, TLR4	Polymorphism	PCR-RFLP	TLR4 Ex4 + 936C>T polymorphism (g.14143C>T; rs4986791) significantly associated with the overall higher risk of GBC in north Indian population	[131]
CYP1A1 (rs2606345)	Polymorphism	TaqMan assay	Associated with increased risk of biliary tract cancer in Chinese populations	[136]
TP 53	Point mutation/loss of heterozygosity	PCR/RFLP	Found in both early and advanced GBC, associated with pre malignant conditions in Indian, Japanese Chile and bolivian population	[137,173-175]
KRAS	Mutation (codon 12/13)	PCR-RFLP	Seen in early lesions; potential marker for pre-malignant transformation	[176]
CDKN2A (p16)	Promoter methylation or loss of homozygosity	PCR-RFLP	Acquisition of hypermethylation contribute to tumor formation and progression within the chronically inflamed gallbladder at early stage of carcinogenesis	[177,178]
ABCG8	Polymorphism	GWAS/PCR-RFLP	Increased risk of GBC in patients with gall stone disease	[179]
ERCC2, MSH2	-	PCR-RFLP	Associated with early steps of carcinogenesis	-

GBC: Gall bladder cancer; PCR-RFLP: Polymerase chain reaction - restriction fragment length polymorphism; GWAS: Genome-wide association study.

Table 10 Liquid biopsy in the diagnosis of gall bladder cancer

Liquid biopsy	Detection method	Sensitivity and specificity	Implementation barrier in early diagnosis	Ref.
CTC	Flow cytometric detection, nano microfluid chip, immunoaffinity (EpCAM), microfiltration (ISET)	55.6%, 100.0%	Very low detection rate in early stage	[193]
			CTCs in peripheral blood (approximately 1 CTC per 106-10 Leukocytes)
			Large blood volume required to detect CTC
			Short half-life of CTC makes it difficult to analyse
miRNAs	qRT-PCR, microarrays, NGS	80%-90%, 80%-90%	miRNA expression can vary depending on samples	[198,199]
			Delay in processing, and temperature fluctuations can alter miRNA levels
			Low abudance, platform dependency, lack of standardization, high cost
LncRNA	qRT-PCR, microarrays, NGS	84%-100%	Low abundance, delay in processing, and temperature fluctuations can alter miRNA levels	[198,199]
			Platform dependency, lack of standardization, high cost
ctDNA/cfDNA	qPCR, dPCR, ddPCR, NGS, high-throughput quantitative methylation assays	78%-100%, 80%-100%	Separation of ctDNA from the cfDNA (mixture of non-mutant tumor DNA, normal DNA and tumor DNA) is technically challenging	[200,201]
	RBBS		High false negative rate because of tumor heterogenicity
			ctDNA used for genetic testing only 0.01% of tumor
			Limited representation of tumor environment
			No standardization and no universally accepted cutoff for detection
			High cost
			Low availability
Bile as liquid biopsy	qRT-PCR, microarrays, NGS	45.8% and 99.9%	Invasive methods to aspirate bile from GB	[202]
			No standardized methods and cutoff value

ctDNA: Circulating tumor DNA; cfDNA: Cell-free DNA; miRNAs: MicroRNAs; lncRNAs: Long noncoding RNAs; GB: Gall bladder; dPCR: Digital polymerase chain reaction; ddPCR: Digital droplet polymerase chain reaction; NGS: Next-generation sequencing; RBBS: Reduced representation bisulfite sequencing; qRT-PCR: Quantitative real-time reverse transcription polymerase chain reaction; CTC: Circulating tumor cell.

Table 11 Proposed scoring system for further evaluation to identify early gall bladder cancer

Patient and imaging factors	Presence or absence of factor	Score
Area from patients belongs	High Endemic area (Chile, Gangetic belt in India, Pakistan, Bolivia)	3
	Moderate endemic area (Japan, Korea, Latin America)	2
	Low endemic area (United States)
	Non endemic area	1
Middle or old aged female	Yes	2
	No	1
Long standing biliary colicky pain	Yes	2
	No	1
Recent change in character of pain	Yes	2
	No	1
Unexplained weight loss present	Yes	2
	No	1
Any associated risk factors like PSC, PBM, calcific gall bladder	Yes	1 for each
Gall stone size	> 3 cm	2
	< 3 cm	1
Polyp	Polyp with lobular surface, vascular core or hypo-echoic polyp or hypoechoic foci or a polyp size of greater than 1 cm or sessile polyp	2
	Other polyps	1
Mass and texture of gall bladder wall	Mass filling GB	3
	Asymmetrical wall thickness	1
	Loss of layered structure of wall localized GB wall thickening > 4 mm	1
	Normal GB wall	1

GB: Gall bladder; PSC: Primary sclerosing cholangitis; PBM: Pancreaticobiliary maljunction.