Early detection of gallbladder cancer: Current status and future perspectives

Abstract

Gall bladder cancer (GBC) remains a highly aggressive disease, with an overall 5-year dismal survival rate of 15%-20%. Its asymptomatic nature in very early stages and non-specific clinical presentations pose significant challenges to timely detection. Consequently, GBC often presents late, making it one of the most challenging cancers to manage. Surgery offers the best chance for long-term survival; however, only 10% of GBC patients are candidates for upfront resection, with the majority presenting in locally advanced or metastatic stages. Furthermore, GBC is generally resistant to chemotherapy and radiotherapy, limiting the effectiveness of systemic therapy. Therefore, early diagnosis is crucial to offer the best treatment through surgical resection and to improve the outcome. Recent advancements in imaging technologies, biomarker discovery, and molecular diagnostics offer promising avenues for enhancing detection rates. Though non-invasive, most of them lack specificity, and the majority fail as an early diagnostic tool. This review examines the current status of early detection strategies for GBC, addresses the limitations of existing approaches, and explores the newer emerging diagnostic tools and techniques and how they can be exploited in future for its early detection.

Key Words: Gall bladder cancer; Early diagnosis; High risk factors; Radiology; Biomarkers; Liquid biopsy; Artificial intelligence

Core Tip: Gall bladder cancer is an aggressive disease with a dismal prognosis. Surgery offers the best treatment. However, most of the patients present late due to nonspecific symptoms and at a stage where resection is not feasible. Therefore, early detection is crucial for better outcomes. Identifying high-risk features, utilizing various imaging modalities, use of biomarkers, liquid biopsy and newer tools like artificial intelligence can aid in the early diagnosis.

INTRODUCTION

According to global data, gall bladder cancer (GBC) ranks 22^nd in incidence and 20^th in mortality worldwide, making it the most common biliary tract cancer. The age-standardized rate (ASR) for GBC incidence is 1.2 per 100000. The highest reported ASR rates have been observed in subpopulations of Northeastern India and Southern Chile. In 2022, China, India, and Japan had the highest numbers of GBC cases and deaths[1]. It’s estimated that only 10% of GBCs are upfront resectable, and most are at an advanced stage[2]. The five-year survival is only 25%-40% in early-stage GBC and 2% for metastatic GBC[3]. In one of the population-based cohort studies from India, the authors found that 1–year and 3-year survival for GBC was 29.0% (22.6%-35.8%) and 5.4% (2.5%-9.8%), with no 5-year survival[4]. Patkar et al[5] found the 3-year overall survival rates for stages I, II and III were 94.1%, 82.6% and 48.2%, respectively. In a large Japanese study, patients with T1 GBC had an 85.9% 5-year survival rate, while those with T3 tumors and T4 tumors had 19.2% and 14.1%respectively[6]. Most patients with GBC are diagnosed at the locally advanced stage, leading to a poor prognosis. As a result, it’s necessary to diagnose at an early stage. GBC lacks specific symptoms, and the existing biomarkers and diagnostic modalities often fail to diagnose it in the early stage. The nonspecific symptoms of GBC, such as right hypochondrial pain, are indistinguishable from those of gallstone diseases. Furthermore, specific clinical signs such as a palpable mass, hepatomegaly, and jaundice are frequently indicative of the advanced stages of the disease. To further complicate the clinical scenario, a significant proportion of patients may show no symptoms and are diagnosed incidentally.

This underscores the critical need for early diagnosis of this dreaded disease. The lack of nonspecific symptoms results in further delays in diagnosis. Surgical resection offers the most effective treatment; however, as mentioned earlier, only a small percentage of patients present at a stage where resection is possible. No definitive diagnostic techniques or biomarkers exist today to aid in early diagnosis. Early diagnosis and timely intervention in patients who are at high risk of developing GBC is the best strategy to confront this dreadful disease. In this review, we discuss various modalities that can be utilized for the early diagnosis of GBC. We have categorized these methods into distinct sections, including susceptible genetic markers, biochemical and molecular markers, in addition to clinical and radiological features.

SEARCH METHODS

We systematically reviewed published literature on GBC between 1995 and 2024 from online search engines PubMed and MEDLINE using the search terms GBC, early diagnosis, clinical and radiological features, biomarkers, molecular markers, genetic markers, liquid biopsy, and bullion operators like “AND, OR, and NOT”. We included only those publications relevant to early diagnosis. Secondary sources retrieved from these publications were identified through manual searches and assessed for relevance. The results are discussed in detail.

CLINICAL SUSPICION

Identifying the population at high risk of GBC

It is crucial to recognize the risk factors that predispose individuals to GBC. Identifying patients at high risk enables early diagnosis, proactive surgical intervention(cholecystectomy), increased awareness, and better symptom recognition. The various high-risk factors associated with the development of GBC are detailed in Table 1[7-24]. Recent data indicate that the highest GBC incidence rates are found in women from India (21.5 per 100000), Chile (18.1 per 100000), Pakistan (13.8 per 100000), and Ecuador (12.9 per 100000). Females in northern India, particularly in Delhi, have an equal ASR compared to Chile and Bolivia[2]. Numerous studies have indicated that populations living in high-risk areas are associated with an increased likelihood of developing GBC. Mhatre et al[25] observed that individuals born in high-risk regions have 4.82 times greater odds of developing GBC [95% confidence interval (CI): 3.87-5.99] compared to those born in low-risk regions. Moreover, a dose-response relationship was identified between the risk of GBC and the duration of residence in high-risk areas, resulting in an odds ratio of 5.58 (95%CI: 4.42-7.05) along with a significant P value (≤ 0.001). The ASR of GBC in Indian females is similar to that in the Chilean population. So, the population from these high-risk areas should be carefully followed and evaluated promptly if required. Similarly, various studies found an increased risk of GBC in populations residing near Indian rivers - Sutlej, Ganges, Yamuna, and Brahmaputra due to heavy metals and industrial activity contaminations. Chhabra et al[26] found that chromium, lead, arsenic, and zinc were significantly higher in Indian patients compared to Japanese patients.

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.

However, screening all these high-risk populations is practically challenging. Therefore, these groups should raise the threshold of suspicion for GBC when they are symptomatic or have asymptomatic gallstone disease. In asymptomatic patients, the evidence does not support that the presence of gallstones leads to GBC[27,28]. There should be a tailored approach for the workup of both symptomatic patients and asymptomatic patients with high-risk characteristics to carefully evaluate for malignancy and detect early cancer should any suspicion arise on clinical or imaging evaluation.

Signs and symptoms which may be helpful in early diagnosis

GBC often presents with nonspecific signs and symptoms. Patients diagnosed with GBC often present with pain located in the right upper quadrant as well as the epigastric region of the abdomen. Many patients give a history of a change in the character of pain, with previous long-standing intermittent pain becoming continuous, dull aching pain. Pain may be persistent and may not respond to routine analgesics. At times, it may be associated with unexplained weight loss. They may be presented with jaundice and features of gastric outlet obstruction in advanced lesions[2]. Pandey et al[29] found that weight loss was the most common symptom (reported in 201 patients, 99%), followed by loss of appetite (reported in 197 patients, 97%). Other symptoms included lower right abdominal pain (143 patients, 70%), lower right abdominal mass (107 patients, 53%), jaundice (79 patients, 39%), and nausea and vomiting (21 patients, 10%). In the authors’ institute, located in an endemic GBC area, almost all patients presented with weight loss or appetite issues. Therefore, in the absence of specific symptoms, patients with unexplained weight loss or appetite should be evaluated for GBC in endemic regions.

Radiological imaging findings in the early diagnosis of GBC

Ultrasonography (USG) is the most widely used method for evaluating the gallbladder (GB). The most common early finding of GBC is thickening of the GB wall. However, USG does not reliably distinguish between benign and malignant GB wall thickening. In this section, we discuss various imaging features of traditional imaging modalities, including USG, contrast-enhanced computer tomography (CECT), magnetic resonance imaging (MRI), and positron emission tomography (PET) scans, as well as newer modalities such as contrast-enhanced ultrasound (CEUS) and elastography for the early diagnosis of GBC.

Ultrasound: For most patients, USG is the initial investigation they undergo. Therefore, it’s crucial to recognize the features of USG that raise suspicion about GBC. Many patients get diagnosed late due to missed findings or an inability to identify suspicious findings on USG. USG identifies multiple abnormalities, including wall thickness, polyps, associated gallstones, masses, ascites, metastases, liver infiltration, and involvement of adjacent structures. Gallstones are often present along with a mass in 60%-90% of cases[30]. Various USG findings that suggest malignant GB lesions include focal and asymmetric wall thickening, discontinuous mucosa, loss of the layered pattern of the GB wall, high peak systolic velocity in doppler, high shear velocity in elastography, and a mass filling the GB lumen[30]. Gupta et al[31] introduced a GB reporting and data system based on GB wall features observed on ultrasound (US). These features include the symmetry and extent (focal vs circumferential) of involvement, intramural characteristics (such as intramural cysts and echogenic foci), layered appearance, and the interface with the liver. This system, based on risk stratification, recommends six categories (GB reporting and data system: 0-5) of GB wall thickening, indicating a progressively increasing risk of malignancy. Batra et al[32] found that the most frequent finding on US was GB mass replacement, observed in 73% of cases, followed by the presence of both a gallstone and a mass in 54% of cases. Rana et al[33] proposed a sonographic “Cervix Sign” for GB neck malignancy. So, early identification of mass in the GB results in a prompt referral to an expert center. As USG is the most basic investigation, the question arises if it can be used as a screening strategy in high-risk populations like North India, Chile and Pakistan.

CEUS: Kong et al[34] found that a higher rate of CEUS had higher sensitivity than USG in detecting GB malignancy. Xu et al[35] found that focal GB wall thickening, inner wall discontinuity, and outer wall discontinuity are distinctive features of GBC. Figueiredo et al[36] found higher sensitivity of CEUS than MRI in detecting GB cancer. Features in CEUS suggestive of malignancy include non-homogeneous arterial phase enhancement, dotted intralesional vascularity, washout time of less than 40 seconds, and liver parenchymal infiltration.

Contrast-enhanced computed tomography: Contrast-enhanced computed tomography (CECT) is the preferred diagnostic modality for staging the disease. Kalra et al[37] found CECT showed a sensitivity of 72.7%, a specificity of 100%, and an overall accuracy of 85% in assessing the resectability of GBC. In detecting hepatic and vascular invasion by the tumor, computed tomography (CT) findings were in complete agreement with surgical findings. Kumaran et al[38] found that CECT had an accuracy of 93.3% in the staging of GBC. Pruthi et al[39] studied the role of dual-energy CT and found that asymmetrical mural thickening and non-layered mural thickening were observed in all patients on iodine maps, as well as at 80 keV and 140 keV. Iodine maps showed heterogeneous enhancement in all patients. The author concluded that dual-energy CT has a clear benefit in identifying malignant GB thickening. CECT findings that directly indicate GBC include irregular GB wall contours, submucosal edema, localized wall thickening, interruption of the mucosal lining, presence of a polypoidal mass, and evidence of direct invasion into the neighboring organs, etc[40]. The indirect signs of malignancy include biliary obstruction, regional lymphadenopathy, paraaortic lymphadenopathy, and distant metastasis. Corwin et al[41] based on CT findings, classified incidental focal fundal GB wall thickening into six types: type 1 (nodular/pinched intramural low attenuation), type 2 (intramural low attenuation), type 3 (homogeneous enhancement), type 4 (nodular/pinched homogeneous enhancement), type 5 (intramural cystic spaces), and type 6 (hyper-enhancing/heterogeneous enhancement). It was found that types 3 and 6 thickening were malignant.

MRI: Kalage et al[42] found that MRI has higher sensitivity than CT in detecting malignant GB thickening. Han et al[43] proposed a scoring system based on nine MRI features indicative of malignant transformation: Diffuse GB wall thickening, mucosal retraction, loss of mucosal uniformity, presence of gallstones, intramural T2 hyperintensity, intermediate-to-high T1 signal intensity, enhancement pattern, the timing of peak enhancement, and diffusion restriction. This MRI-based scoring model demonstrated outstanding diagnostic accuracy, achieving an area under the receiver operating characteristic curve (AUC) of 0.972, markedly outperforming conventional visual assessment by radiologists. Benign GB wall thickening shows hyperintensity on T1-weighted and T2-weighted images. The malignant thickening exhibits moderate hyperintensity on T2 imaging, presenting a papillary appearance along with diffusion restriction[44]. On contrast-enhanced MRI, benign GB wall thickening typically exhibits gradual enhancement, while malignant thickening shows rapid early enhancement as a result of neovascularization[45]. Presence of intramural foci and small cystic changes suggestive of adenomyomatosis. Similarly, the presence of hypoechoic or hypoattenuating nodules in the GB wall is suggestive of xanthogranulomatous cholecystitis (XGC). Jung et al[46], based on MRI findings, classified the layered pattern of the thickened wall into four types: type 1 exhibits two layers, with a thin hypointense inner layer and a thick hyperintense outer layer; type 2 presents two layers with ill-defined margins; type 3 features multiple hyperintense cystic spaces in the wall; and type 4 displays diffuse nodular thickening without layering. Chronic cholecystitis corresponds to type 1, acute cholecystitis aligns with type 2, adenomyomatosis is classified as type 3, and GB carcinomas correspond to type 4. Each of the four-layered patterns showed a PPV of at least 73%, a sensitivity of 92% or higher, and a specificity of 95% or higher.

PET scan: (18)F-fluorodeoxyglucose (FDG) uptake in GB may result from both inflammatory and cancerous conditions. PET-CT is usually indicated for evaluating distant metastasis but can also be used when there is GB wall thickening and any diagnostic dilemma with conventional imaging methods. Both the GB wall thickness and the standardized uptake value (SUVmax) are important in diagnosing GBC and play complementary roles in diagnosis[47]. Benign conditions usually have a lower SUVmax value compared to malignant lesions[48]. Ramos-Font et al[47] found that at a cutoff value of 3.62, diagnostic accuracy was 95.9%. Gupta et al[49] reported a sensitivity and specificity of 92% and 79%, respectively, at a cutoff value of SUVmax of 5.95 and 94% and 67% when using a cutoff value of 8.5 mm.

In Table 2[50-68], we have summarized the various imaging features of malignant GB lesions from various studies. In addition to diagnostic values, other factors such as cost and the availability of various imaging modalities also influence detection in resource-poor countries like India. USG is the most widely available modality, being both easily accessible and the least expensive option; it is the first investigation that most patients undergo. Therefore, it is essential for radiologists and clinicians to understand the various features indicative of malignant changes. Table 3[45,55,57,60,61,69-82] summarizes the sensitivity, specificity and limitations of conventional imaging modalities in the detection of early GBC.

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.

NEWER IMAGING MODALITIES FOR THE DIAGNOSIS OF GBC IN THE EARLY STAGE

Traditional imaging modalities, like USG, CT, and MRI, are often used when there are obvious symptoms, and the disease is in an advanced stage. In addition, they lack the specificity to differentiate between benign and malignant lesions. The newer imaging modalities may be useful tools for early diagnosis.

Real-time elastography (RTE) differentiates tissues according to their rigidity, proving to be a promising method for distinguishing malignant from benign tissue. Kapoor et al[83] found that, at a cut-off value of 2.7 m/second, elastography showed an overall accuracy of 92.8% with sensitivity and specificity of 100% and 91.3%, respectively, for diagnosing GB carcinoma. RTE leveraging acoustic radiation force impulse is effective in distinguishing benign from malignant GB masses. This technique relies on the concept that malignant tissues exhibit significantly greater stiffness, resulting from higher cell density, in contrast to tissues affected by chronic inflammation and fibrosis. Teber et al[84] studied the RTE of GB polyps and discovered that benign GB polyps on successive RTE images exhibited a high-strain pattern. Soundararajan et al[85] found that the mean shear wave elasticity of the uninvolved region in GBC was significantly higher than that of benign conditions like gallstone disease and controls. The Author found that sensitivity was 91.3%, specificity was 83.5%, with an AUC of 0.927.

Aside from detecting malignant GB conditions, imaging also helps to differentiate early from late GBC. Some of the imaging findings are specific (Table 4) for the early detection of GBC. Based on the imaging features above, in the next section, we describe various conditions, such as polyps, thick-walled GB, XGC, and calcified GB, that are at risk of developing cancer or are associated with cancer. We have delineated important features that differentiate them from malignancy.

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.

Early identification of malignant GB polyps

GB polyps can be divided into neoplastic and non-neoplastic polyps. Neoplastic polyps are adenomas and adenocarcinomas, and non-neoplastic polyps are cholesterol polyps, inflammatory polyps and adenomyomatosis, etc. Among newer modalities, endoscopic US (EUS) can distinguish the two-layered architecture of the GB wall and offers superior resolution for detecting small polypoid lesions. Choi et al[86], based on five parameters (layer structure, the margin of the polyps, echo patterns, and stalk and polyp numbers), proposed a scoring system to detect malignant polyps. Similarly, Sadamoto et al[87], based on EUS, proposed a formula: maximum diameter (in millimeters) + internal echo pattern score (heterogenous = 4, homogenous = 0) + hyperechoic spot (5) to differentiate benign vs malignant polyps. The PET scan can also be used to diagnose malignant GB polyps. Lee et al[88] found that the SUVmax of GB polyp and the ratio of SUVmax of polyp mean SUV of the liver (GP/L ratio) were high predictors of malignancy. Another study found that delayed (18) F-FDG PET is more helpful than early (18) F-FDG PET for evaluating malignant lesions because of heightened uptake within the lesion and improved contrast between the lesion and surrounding tissue[68]. Various other commonly used investigations that were used to differentiate benign from malignant GB polyps are described in Table 5[52,74,89-100].

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.

Thick-walled GB

Many cases of GBC present with only wall thickening without any mass or associated stone. Wall thickening usually refers to a thickness greater than 3 mm. In Table 6, we have summarized the findings that differentiate benign from malignant wall thickening from the literature.

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.

Pancreatic-biliary malunion

Various studies have found that pancreaticobiliary reflux is a significant risk factor for biliary tract cancer in patients with pancreatic-biliary malunion (PBM). Kamisawa et al[100] found that among 49 patients with PBM, without biliary dilatation, GBC was identified in only 33 cases. Misra et al[101] also found that an abnormally long common channel (greater than or equal to 8 mm) was seen more frequently in carcinoma of the GB compared with normal subjects and patients with gallstones. Sugiyama et al[102] also found a similar finding that PBM without biliary dilatation is associated with an increased risk of malignancy. Therefore, when identified, it should be kept in mind that these patients have a very high risk of malignancy and carefully further evaluated.

Porcelain GB

The presence of mural calcification is considered premalignant, and previous studies have documented a malignancy rate of 7%-60%[103]. Recently, however, it has become clear that not all patients with porcelain GBs are pre-malignant. Stephen et al[104] noted that there are two categories of calcified GBs: One exhibiting complete intramural calcification and the other showing selective mucosal calcification. The likelihood of cancer developing in a GB with selective mucosal wall calcification is about 7%, while none of the patients with diffuse intramural calcification have been found to develop cancer. According to current evidence, the incidence of GBC, in general, is around 2% to 8%, particularly among those with partial presence of calcification adherent to intact mucosa. There appears to be no GBC risk in specimens where the GB wall is replaced with calcium, and there is no intact mucosa[103,105].

XGC

XGC is a malignant masquerade of GBC. This is a long-lasting inflammatory condition of the GB, linked to GBC in 8.5% to 30.5% of instances[106-108]. The association is crucial because the presence of both lesions in the same specimen can lead to overlooking the carcinoma entirely. Therefore, it is essential to identify the XGC pre-operatively. The Presence of hypoechoic nodules or bands in the thickened wall is regarded as a characteristic finding of XGC. Hypoechoic nodules on sonography have been observed in 15% and 73% of cases by Parra et al[109] and Kim et al[110], respectively. CECT commonly shows symmetrical diffuse thickening in XGC, though 22.2% of cases display asymmetrical thickening. The intramural nodules were detected in 85.7% and 61.1%, respectively, by Zhao et al[111] and Goshima et al[112]. XGC affects the pathology affecting the GB wall, where the mucosal surface remains either intact or slightly damaged. In contrast, GB carcinoma develops from the GB epithelium, leading to significant mucosal disruption in most cases. Mucosal disruption in the XGC mucosal lining is more likely to indicate malignant change. Enhancement of the luminal surface (LSE) is characterized by continuous mucosal lines or mucosal lines showing focal breaches linked to XGC. LSE was noted in 70% of cases by Shuto et al[113]. LSE and preservation of the epithelial layer point towards benign disease[113]. So, it’s important to identify the subset of the population with XGC with a risk of malignant features for early evaluation.

ROLE OF BIOMARKERS IN EARLY DIAGNOSIS OF GBC

Biomarkers

Recent research has focused on refining the diagnostic and prognostic utility of existing biomarkers, developing new biomarkers such as liquid biopsy tools, and enhancing imaging techniques that can more effectively distinguish between benign and malignant pathologies. In this section, we have described various markers and their clinical utility in the early diagnosis of GBC.

Serological markers: Previous studies have failed to demonstrate the utility of biomarkers like carbohydrate antigen (CA) 19-9 (CA19-9), carcinoembryonic antigen (CEA), CA125, and CA242 in diagnosing GBC due to low sensitivity and specificity. Wang et al[114] studied CEA, CA125, CA242, and CA19-9 and found that CA19-9 had the highest sensitivity (71.7%), while CA242 had the highest specificity (98.7%). Furthermore, inflammatory disorders did not impact CA242 levels. The author also found that a combination of CA19.9, CA242, and CA125 had the highest diagnostic accuracy (69.2%). In Table 7, we have described the diagnostic utility of serum biomarkers in GBC[115-122].

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.

Among some newer biomarkers, Oshikiri et al[123] found high receptor-binding cancer antigen expressed on SiSo cells (RCAS1) expression in 32 of the 46 GBC specimens (70%) but not in cases of cholecystitis, adenomyomatosis and adenomas. However, the author found that RCAS1 expression was believed to promote tumor progression and to be a relatively late event in GB carcinogenesis, thereby undermining the role of RCAS1 in the early-stage diagnosis. Giaginis et al[124] also suggested the role of RCAS1 expression only in tumor prognosis, not in early diagnosis. Koopmann et al[125] evaluated the performance of Mac-2 binding protein and its ligand, galectin-3, as diagnostic markers of biliary carcinoma and found that it can differentiate between malignant and benign biliary diseases with sensitivity and specificity of 69% and 67%, and the AUC was 0.70. Ghimire et al[126] evaluated the levels of cytokeratin-19 fragment antigen 21-1 (CYFRA 21-1) and CA19-9 in 61 patients diagnosed with biliary tract cancers, most notably GBC as the prevalent type of group. They found that CYFRA 21-1 had a sensitivity of 80.3%, and CA19-9 had a sensitivity of 68.9 % for detecting biliary tract cancer. More recently, Huang et al[119] discovered that CYFRA 21-1 at a cut-off value of 3.27 (ng/mL) outperformed CEA and CA19-9 in diagnosing GB carcinoma and intrahepatic cholangiocarcinoma.

Susceptible genetic markers: The pathophysiology of GBC is multifactorial and influenced by several risk factors, including predisposing conditions, gallstones, chronic cholecystitis, porcelain GB, GB polyps, obesity, and exposure to toxic minerals. Genetic predisposition also plays a crucial role, as highlighted in various studies. Somatic mutations are present in 90.0 % of GBC[127]. This highlights the importance of detecting somatic genetic mutations in GBC in suspected early-stage disease, as it may aid in identifying high-risk individuals and potential therapeutic targets. By analyzing genetic factors, researchers have identified critical genes and specific patient subgroups at increased risk of developing GBC, thus facilitating early detection and timely intervention. Genome-wide association studies have identified numerous genetic variants associated with a heightened risk of developing GBC. These molecular genetic markers are based on histological specimen analysis and may play a significant role in early diagnosis. Various genetic markers are described in Table 8[128-171].

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]

Barreto et al[172] proposed a genetic model similar to the adenoma-carcinoma sequence for GBC, illustrating how genetic changes shift from chronic inflammation to malignant transformation (Figure 1). Genetic mutations at different stages of pathogenesis, such as from inflammation to carcinoma in situ, can be used to detect GBC at an early stage. These mutations can be investigated in high-risk populations for the early diagnosis of GBC, such as individuals with chronic cholecystitis of long duration and from the high GBC belt, and also for screening purposes. In addition to diagnosis, these markers can be used for various diagnostic and therapeutic applications. In Table 9[131,136,137,149,168,173-179], we have summarized some of the genes that have demonstrated clinical utility in the early diagnosis of GBC in small cohort studies and can be considered for testing in high-risk conditions, as outlined in section 1 for early identification and risk stratification.

Figure 1 Pathogenesis of gall bladder cancer-biomarkers working at different stages. GB: Gall bladder.

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.

LIMITATIONS OF MOLECULAR MARKERS

While identifying genetic markers, such as various genes mentioned above, has diagnostic utility for early identification of high-risk groups, there are several limitations to their use as diagnostic tools. These include the absence of a universally accepted genetic panel for GBC diagnosis, the high cost and requirement for molecular laboratories, low awareness and accessibility in resource-poor settings, Genetic predisposition varies widely among populations and a lack of extensive data regarding real-world clinical utility.

Liquid biopsy tools in early diagnosis: Over the last decade, the concept of liquid biopsy has evolved, which involves detecting and analyzing circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), and microRNA (miRNA), circulating tumor cells (CTCs), and extracellular tumor vesicles released into blood and other body fluids like bile and urine[180]. One method in liquid biopsy, which provides information about the tumor through CTCs, circulating tumor DNA, miRNAs, and exosomes released by the tumor, collectively known as genomics. Another method is proteomics/metabolomics, which reflects specific conditions in the tumor. The third method is transcriptomics, which involves the study of RNA transcripts. Genomics uses various genes, DNA, mRNA, etc., for analysis via quantitative real-time polymerase chain reaction (PCR), digital PCR, digital droplet PCR, polymerase chain reaction-restriction fragment length polymorphism, and next-generation sequencing (NGS). Proteomics measures various proteins derived from tumor cells via immunohistochemistry and fluorescence in situ hybridization methods (Figure 2)[181].

Figure 2 Various liquid biopsy tools. NGS: Next-generation sequencing; PCR: polymerase chain reaction; FISH: Fluorescence in situ hybridization; CAGE: Cap analysis of gene expression; PRO-cap: Precision RNA on-cap; PAR-CLIP: Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation; SMART: Simple Modular architecture research tool; cfDNA: Cell-free DNA; ctDNA: Circulating tumor DNA.

The role of liquid biopsy as a non-invasive diagnostic biomarker compared to tissue biopsy has recently emerged. Assessing blood, urine, and saliva is simpler than evaluating tissue samples, leading to a faster process and simpler method for diagnosis[175,176]. Liquid biopsy, although highly useful in metastatic testing, has a less studied role in early diagnosis and faces many constraints. Tumors in early stages exhibit a low variety of alleles, resulting in diminished diagnostic accuracy for their detection variants[182]. Similarly, ctDNA utilized in genetic testing constitutes a minuscule fraction of the tumor, occasionally dropping to as low as 0.01%. CTC are typically present in low quantities in early-stage tumors, making their detection and quantification particularly challenging compared to late-stage or metastatic GBC. Another constraint involves the higher blood volume needed for CTC detection. Moreover, elements like sample integrity, the fleeting nature of CTCs, and interference from circulating nucleic acids can greatly impact result reliability[127]. Moreover, sample integrity, the short lifespan of CTCs, and the influence of circulating nucleic acids impact the outcome results. As a result, the role in early diagnosis is not well established because of the low sensitivity of liquid biopsy methods.

ctDNA/cfDNA

Increased levels of cfDNA have already been reported in various malignancies, including colon, head and neck, gastric, pancreatic, and hepatocellular cancers[183]. Kumari et al[184] found that cfDNA was significantly higher in cancer patients versus cholecystitis and control groups, with a cut-off of > 218.55 ng/mL showing 100% sensitivity (95%CI: 89.6-100; P < 0.001) and specificity (95%CI: 80.3-100; P < 0.001) for GBC compared to controls. For GBC versus patients with cholecystitis, the cut-off of > 372.92 ng/mL provided 88.24% sensitivity (95%CI: 72.5-96.6; P < 0.001) and 100.00% specificity (95%CI: 84.4-100.0; P < 0.001) for cholecystitis. Another study by Kumari et al[185] studied the role of cfDNA as a liquid biopsy in the diagnosis of GBC utilizing levels of long DNA fragments (ALU247) derived from tumor necrosis, short apoptotic fragments (ALU115) denoting total cfDNA and cfDNA integrity denoting the ratio of ALU247 and ALU115. They found that the sensitivity and specificity of ALU247 in discriminating GBC from controls were highest, with a sensitivity, specificity and diagnostic accuracy of 80.0%, 86.1% and 82.2%, respectively. Shen et al[186] studied ten biliary tract cancer patients, four with GB carcinomas and six with cholangiocarcinomas, aiming to identify individual actionable mutations derived from bile cfDNA using targeted deep sequencing. The study found high sensitivity (94.7%) and specificity (99.9%) when comparing bile cfDNA and tumor DNA for single nucleotide variation/insertion and deletion. For copy number variation, sensitivity was 75.0%, with a specificity of 98.9%. The percutaneous or operative method of bile sampling showed no significant difference in single nucleotide variation/insertion and deletion or copy number variation detection sensitivity. Mishra et al[187] found that NGS-based cfDNA mutation profiling can be used to diagnose GBC before surgery and guide treatment decisions. Currently, ctDNA/cfDNA has no proven role in early diagnosis, but newer tools and technology, in future research, may come with its usefulness in early GBC.

Epigenetic alternation

Since ctDNA/cfDNA sensitivity is low in early-stage cancer patients, large-scale epigenetic changes that are specific to tissue and cancer types may be more effective in detecting and classifying cancers in early-stage disease. Various studies have also demonstrated that epigenetic alterations in cfDNA, which are tissue and cancer-type specific have a more remarkable ability to detect and classify cancers in patients with early-stage disease. NGS can detect these epigenetic changes easily[188-190].

CTCs

It is rare to find CTCs in peripheral blood (approximately 1 CTC per 106-10 leukocytes); however, their detection and quantification may identify cancer patients at high risk of metastasis and indicate tumor progression rather than early diagnosis[127]. In a study by Awasthi et al[191], CTC was found in 92.59% (25/27) of GBC cases, with a median count of 4 CTCs/ml (range: 0-20), and a sensitivity of 92.6% and specificity of 91.7%, respectively. Yan et al[192] reported an overall CTC detection rate of 19.80% among 101 GBC patients, with CTC positivity observed in 61.54% (8 out of 13) of patients in the inoperable group and 13.64% (12 out of 88) in the surgical group. Due to the extremely rare peripheral smear, the role of CTC in early detection is still to be proved and only helps in prognostication rather than early diagnosis.

Long noncoding RNAs

Various long noncoding RNAs and miRNA are epigenetic changes that can serve as early diagnostic markers. These RNAs influence cancer-related signalling pathways in GBC, including WNT/-catenin, phosphatidylinositol 3-kinase/protein kinase B, epidermal growth factor receptor, Notch, mammalian target of rapamycin and TP53[127]. Around 30% of global gene expression is regulated by miRNA, and abnormal expression of various miRNAs has been linked to numerous human cancers[193]. Their high stability enables detection in various body fluids, making them promising biomarkers for early diagnosis and prognosis[194]. Additionally, miRNAs, short non-coding RNAs, play a role in tumor initiation[195]. Yang et al[196] found that suggested miRNAs could be utilized in diagnosing and screening GBC, with their study revealing that the sensitivity of miR-4433a-3p was the highest at 96.67%, and the specificity of miR-551b-3p also reached 96.67%. Srivastava et al[197] studied the role of tumor-associated miRNA expression for the early diagnosis of GBC and found that serum levels of miR-21 and miR-182 were upregulated, while miR-130, miR-146, and miR-182 were downregulated in GBC compared to normal cholecystitis. They discovered that miR-1 exhibited the greatest sensitivity at 85.71%, while miR-21 demonstrated the highest specificity at 92.73%. Mishra et al[198] studied the role of long noncoding RNAs in early diagnosis of GBC and found that the expression of serum hox antisense intergenic RNA, antisense non-coding RNA in the ink4 locus and H19 were upregulated, and colon cancer associated transcript 1 and maternally expressed 3 were downregulated in GBC compared to normal control and cholecystitis. Ueta et al[199] studied the role of miRNAs in serum extracellular vesicles in early diagnosis of GBC and found that serum extracellular tumor vesicles miR-1246 were significantly upregulated and miR-451a was downregulated, respectively, in GBC patients (P = 0.005 and P = 0.001).

Although the field of research in liquid biopsy is expanding, there are still some technical limitations in adopting liquid biopsy in the diagnosis of GBC. The isolation of ctDNA from cfDNA - which comprises a mixture of normal DNA, non-mutant tumor DNA, and tumor DNA - can be technically challenging. The ctDNA utilized for genetic testing constitutes a minimal fraction of the tumor, occasionally amounting to as little as 0.01%. CTC are difficult to detect in early tumors and is seen more commonly in metastatic settings. A larger blood volume is also required for CTC detection, and it represents a minor tumor environment. There is a need to enhance the diagnostic tests for liquid biopsy for the early diagnosis of GBC. Table 10 summarizes the current status of liquid biopsy in GBC[193,198-201].

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.

Bile as a liquid biopsy tool

In biliary tract cancer, bile is in direct contact with the tumor, so it’s expected that protein, genetic material, and tumor cells are continuously shed in the bile, which helps in the detection of cancer. However, various methods to sample bile are invasive, and there is also a small risk of needle tract seedings. Various methods are percutaneous approaches like aspiration, Percutaneous transhepatic cholangiography, Percutaneous cholecystostomy, and various endoscopic approaches are Endoscopic retrograde cholangiopancreatography, Endoscopic naso-biliary drainage, EUS-guided aspiration, etc. Bile can also be used to detect cfDNA and ctDNA. In GBC, Kinugasa et al[202] using NGS, analyzed the cytologic and biliary ctDNA. They found sensitivities of 45.8% and 58.3%, respectively, with an agreement rate of 87.5%, indicating a higher sensitivity of bile ctDNA in GBC.

Bile spectroscopy

Nuclear magnetic resonance spectroscopy (MRS) and liquid chromatography-tandem mass spectrometry are commonly used to differentiate bile composition, aiding in distinguishing benign conditions from malignant ones. However, research is limited, and nuclear magnetic resonance could be further investigated for the early diagnosis of GBC[203].

FUTURE PROSPECTIVES

Many new innovations are upcoming in the field of diagnostics, and they will be useful in early detection soon.

Alternative splicing

Alternative splicing may result in oncogenic protein isoforms or the absence of tumor suppressors. Around 65% of human genes are alternatively spliced. Splice variants of key genes involved in cell cycle regulation, apoptosis, and immune evasion can drive GBC progression. Some splice variants may result in epithelial-mesenchymal transition and give rise to metastasis (CD44, FGFR2). Some splice variants confer resistance to chemotherapy and targeted therapies by altering drug targets or apoptotic pathways. Circulating splice variants of RNA in blood or bile may lead to the early detection of GBC. Splice variants of some candidate genes like TP-53, BCL-X, and MCL 1 may aid in the early detection of GBC[193,204].

Tumor microenvironment markers

The tumor microenvironment, which consists of tumor cells, inflammatory cells, epithelial cells, and stromal cells, plays a crucial role in the pathogenesis of GBC and helps in the early detection of GBC. Various tumor-associated macrophages like CD163⁺ tumor-associated macrophages, T cell exhaustion markers like programmed death-ligand 1, cytotoxic T lymphocyte-associated antigen-4, various cytokines like interleukin 10, transforming growth factor and hypoxia and angiogenesis markers such as hypoxia-inducible factor 1-alpha and vascular endothelial growth factor, as well as endothelial markers like CD31 and angiopoietin-2, can facilitate the early detection of GBC. Additionally, cancer-associated fibroblasts and extracellular matrix markers such as alpha-smooth muscle actin, fibronectin, matrix metalloproteinases-9, and matrix metalloproteinases-2 can also be utilized for early detection and prognostication. These potential markers can be detected through liquid biopsy, enzyme-linked immunosorbent assay, multiplex assays, and AI-based prediction models for early detection[193,205].

MRS

MRS has emerged as a valuable technique for assessing biochemical changes linked to cancer tissues. MRS enables a non-invasive evaluation of metabolite levels in tissues, shedding light on cellular processes frequently disrupted in cancers such as GBC[206]. The utilization of MRS to assess choline levels in GBC represents a significant research focus. MRS allows for a non-invasive evaluation of metabolite concentrations in tissues, such as choline, which is part of cell membranes. elevated choline levels are linked to increased cell proliferation and malignant transformation, suggesting that MRS could serve as a valuable tool for identifying higher choline levels as a biomarker for GBC detection. There are not many advances in the use of MRS in GBC, but the use of MRS alone or in combination with other modalities may enhance the early detection of GBC[207].

Artificial intelligence

Artificial intelligence (AI)-based algorithms can be integrated into CT scans to analyze GBC. Deep learning AI can examine CT scans and deliver accurate diagnoses. Deep learning algorithms have demonstrated exceptionally promising results, significantly improving the efficacy and accuracy in early diagnosis of GBC[208]. Fujita et al[209] achieved accuracy rates of 98.35% by utilizing deep neural networks and MobileNet architectures. Radiomics analysis and computer-aided diagnosis programs can also be used to improve diagnostic efficacy[210]. AI models based on demographic parameters and molecular biomarkers can be used to detect GBC at an early stage. AI applications in GBC detection encounter limitations such as dataset biases that impact accuracy and interpretability issues that affect clinician trust. Essential steps include extensive validation, enhancing interpretability, and ensuring seamless integration into clinical practice workflows[208].

The authors proposed a Scoring system for the further evaluation of patients to identify early GBC. Based on demographics, clinical features, and imaging findings, the authors propose a scoring system to further evaluate patients for early detection of GBC. This scoring system (Table 11) aims to ensure that individuals presenting with biliary colic and specific imaging findings are considered for additional evaluation.

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.

Based on the scoring system, patients can be divided into three groups: Very high risk (> 15), high risk (11-14), intermediate (7-10), and low risk (6). If suspicion is very high, high, or intermediate, it should be carefully evaluated to detect early GBC. The scoring system was developed based on clinical reasoning and relevant literature. These scores are not statistically derived and currently serve as a conceptual framework. The limitations of using arbitrarily assigned scores include potential bias, over- or underestimation of certain clinical or radiological features, and the lack of evidence-based weighting. However, prospective studies are required to validate the proposed scoring system.

CONCLUSION

Despite several advances in treatment modalities for GBC, the early detection rate remains very poor and presents a challenge for surgeons. Most GBCs are identified at a late stage due to non-specific signs and symptoms. So, it’s important to identify high-risk populations, such as patients with GB wall thickening or polyps who have a long duration of symptoms and are from high GBC belt areas, along with those experiencing weight loss or appetite changes, for further evaluation to detect early GBC. Currently, the diagnosis is mainly based on a high index of clinical suspicion and on imaging modalities. So, every surgeon and radiologist should be aware of the early imaging features of GBC, such as asymmetrical wall thickening, loss of layering on USG, discontinuity, and irregularity of the innermost enhancing layer CECT. Judicious use of newer imaging tools, molecular genetics, and biomarkers - alone or in combination - is essential for detecting early disease. Single diagnostic tools are still not very effective in early diagnosis; a multimodal approach is necessary in suspected cases. Further research is required to validate and standardize the real-world applicability of the newer imaging modalities, markers, tools, and technology in the early detection of GBC.