Contrast‐enhanced ultrasound for the diagnosis of hepatocellular carcinoma in advanced chronic liver disease

Abstract

This is a protocol for a Cochrane Review (Diagnostic test accuracy). The objectives are as follows:

To assess the diagnostic accuracy of contrast‐enhanced ultrasound (CEUS) for the diagnosis of HCC of any size and at any stage in people with chronic advanced liver disease.

Background

Hepatocellular carcinoma (HCC) is the most common primary liver neoplasm. Usually, it develops in the setting of chronic liver disease. It represents the third most common cause of death from cancer worldwide, with exceedingly high rates in East and Southeast Asia, several areas in Africa, and southern Europe (Bertuccio 2017). Since the early 2000s, HCC has been one of the few cancers showing unfavourable trends in several areas of the world including Europe, and North and Latin America (Bralet 2000; Hashim 2016; Ryerson 2016). In Europe and North America, the incidence and mortality rates have increased since the mid‐2000s (Bertuccio 2017). Mortality rates are reported to be two‐ to five‐fold higher in Japan, Hong Kong, and Korea than in most European countries, and, in the Americas, the reported trends are downward (Bertuccio 2017). Most common risk factors include liver cirrhosis; severe liver fibrosis; chronic infections with hepatitis B and C; heavy alcohol intake; tobacco use; overweight; diabetes; metabolic syndrome; aflatoxins (poisonous carcinogens produced by Aspergillus flavus and Aspergillus parasiticus, which grow in soil, decaying vegetation, hay, and grains); and non‐alcoholic fatty liver disease (Yang 2011; Bosetti 2013; Bosetti 2014; Stanaway 2016; Bertuccio 2017), although cases of HCC without known risk factors have been reported (Bralet 2000; Young 2012).

Clinically, HCC is frequently diagnosed in the late stage because of the absence of specific symptoms, other than those related to chronic liver disease. Less than 20% of patients are eligible for curative treatment including liver resection, transplantation or ablation due to advanced tumour stage, liver dysfunction, or shortage of liver donors (Davila 2012). Furthermore, curative treatment options are unfeasible in most patients due to severe clinical deterioration at the moment of diagnosis confirmation or the inaccuracy of preoperative clinical evaluation and staging procedure (or both).

Despite the poor initial prognosis (with an overall ratio of mortality to incidence of 0.95) (Ferlay 2012), a five‐year survival rate of more than 50% can be achieved if the HCC is detected at an early stage (Forner 2012). According to the modified Barcelona Clinic Liver Cancer (BCLC) staging system (Llovet 1999; Forner 2018), only people with very early‐ or early‐stage HCC are eligible for curative treatment. Therefore, accurate and early diagnosis of HCC is of high importance.

Prior to advancements in medical imaging, biopsy and cytological examination of the liver specimen were used to make a definitive diagnosis of HCC (Tao 1984). With the development of advanced imaging, the improvement of imaging techniques, and the definition of accurate criteria for the diagnosis of HCC, the need for invasive tests such as liver biopsy was reduced (Forner 2008; Sangiovanni 2010; Manini 2014; LI‐RADS 2017). Concerns have also been raised regarding the risk associated with biopsy of tumour seeding, bleeding, and rate of false‐negative results (Silva 2008; Pomfret 2010). As of 2019, liver biopsy is reserved for lesions with atypical appearance and when imaging results are either equivocal or for lesions in a non‐cirrhotic liver (Bruix 2011; Omata 2017; EASL 2018; Heimbach 2018).

Due to development of microbubble contrast agents, contrast‐enhanced ultrasound (CEUS) has gained increasing interest and offers the potential for ultrasound to show enhancement patterns in liver lesions (Niu 2013). Dynamic CEUS images are obtained similarly to contrast‐enhanced computer tomography (CT) and magnetic resonance imaging (MRI): depending on the time of image acquisition after intravenous contrast injection, the study differentiates arterial and portal venous phases in which sonographic hallmarks for HCC (such as arterial hyperenhancement and subsequent washout appearance) are investigated (Chung 2015; LI‐RADS 2017). Unlike the contrast agents used in CT and MRI, ultrasound contrast agent is a purely intravascular agent, and, therefore, highly accurate in detecting tumour angiogenesis (Schirner 2004). However, due to the nature of the contrast, CEUS does not depict the HCC capsule that is another hallmark in liver lesion characterisation on CT and MRI (LI‐RADS 2017). Advantages of CEUS over CT and MRI include real‐time imaging, use of contrast agents that do not contain iodine and are not nephrotoxic, possible multiple injections of contrast in the same examination, safety, practicality, no risk of nephrotoxicity, no ionising radiation, and short time of image acquisition. However, CEUS is not recommended for disease staging or assessment of treatment response and the adequacy of the examination depends on the liver window and expertise of the operator (LI‐RADS 2017).

Despite the advantages, the use of CEUS in the diagnostic algorithm for HCC remains controversial with disagreements between relevant guidelines (Omata 2017; EASL 2018; Heimbach 2018). Previous systematic reviews have assessed the performance of CEUS in detecting HCC and they have included different studies and yielded different results. Most of these reviews are comparative reviews that compare two or more tests (CEUS, CT, MRI) and address a wider question, that is, the diagnosis of any focal liver lesions not only HCC, but also benign tumours and metastases (Westwood 2013), or a narrower question, that is, only small HCC, with a diameter less than 2 cm (Niu 2013; Deng 2016). Assessment of methodological quality and definition of inclusion criteria, type of studies, and reference standards are often inconsistent. Furthermore, these reviews did not put the index tests into context and did not define clearly their role. Instead they compared all the available tests as they were used simultaneously (Hanna 2016; Huang 2017; Zhang 2017).

The aim of this systematic review and meta‐analysis is to use Cochrane methodology to determine the accuracy of CEUS for the diagnosis of HCC of any size, as well as the diagnosis of resectable HCC in people with chronic advanced liver disease.

Target condition being diagnosed

Hepatocellular carcinoma

HCC is the most common primary liver cancer which occurs mostly in people with chronic liver disease. The incidence of HCC increases in people with hepatitis B and C, alcohol use, and non‐alcoholic fatty liver disease, and people with liver cirrhosis of various aetiology (Bruix 2011). There is no definite threshold in the definition of lesion size, although literature tends to classify lesions with a diameter of 2 cm or less as small (Hussain 2002; Choi 2014; Park 2017).

In clinical practice and according to pertinent guidelines, multiphasic CT or MRI with intravascular contrast application allow for a highly accurate diagnosis of HCC without an invasive biopsy. The diagnosis of HCC is usually based on cross‐sectional CT or MRI features: focal liver lesions which show non‐rim‐like hyperenhancement in the arterial phase, subsequent non‐peripheral washout appearance, and capsule appearance (LI‐RADS 2018). Liver histology is required only for undefined lesion at CT and MRI (Omata 2017; EASL 2018; Heimbach 2018).

Several staging systems for HCC have been proposed and developed; however, there is no globally applicable staging system (Kinoshita 2015). Among different protocols, the modified BCLC staging system has a notable feature of treatment recommendations for each stage based on the best treatment options currently available (Llovet 1999; Llovet 2003; Llovet 2008; Forner 2018). It is comprised of four elements: tumour extension, liver functional reserve, physical status, and cancer‐related symptoms. According to BCLC staging, only people with early‐stage HCC are eligible for curative treatment such as surgical resection or percutaneous treatment.

Orthotopic liver transplantation is reserved for people with decompensated cirrhosis, and is considered a definite curative treatment for HCC. The early experience with orthotopic liver transplantation for HCC in the 1980s included initial poor five‐year survival and high recurrence leading to orthotopic liver transplantation being contraindicated in HCC (Yokoyama 1990). In 1996, specific criteria, known as the Milan criteria, were developed for HCC patient selection (Mazzaferro 1996). These criteria have been repeatedly validated and their value is considerable (EASL 2018). With their implementation, overall five‐year survival of postorthotopic liver transplantation patients exceeded 70% (Mazzaferro 2011). The criteria for patients eligible for orthotopic liver transplantation include single HCC lesion with diameter of 5 cm or less; or up to three HCC lesions, each with diameter of 3 cm or less; no vascular invasion; and no extrahepatic involvement (no metastasis).

Index test(s)

CEUS is an advanced form of ultrasound examination in which images are acquired using intravenously injected microbubble contrast agent with optimised technology required for contrast visualisation. Contrast agent particles are small bubbles similar in size to red blood cells. These microbubbles contain low soluble gases encapsulated into a biocompatible membrane which may have variable composition of lipids, proteins, biopolymers, or a combination of these.

Like in any other contrast‐based imaging procedure, the CEUS examination consists of a bolus administration of contrast media through a superficial peripheral vein. Due to their extremely small size, the microbubbles pass through the pulmonary circulation and then disseminate into the systemic circulation through the arterial bloodstream. The contrast agent remains in the bloodstream for four to five minutes. There is also a parenchymal phase at the level of the liver and spleen because the contrast agent is captured by the reticuloendothelial system or it becomes adherent to the hepatic sinusoid (or both). The gas used for CEUS is eliminated through the airways 10 to 15 minutes after administration, while the substances that form the membrane are eliminated through the kidneys or metabolised by the liver. The use of CEUS in the examination of the liver has special features due to its double vascularity: through the portal vein (two‐thirds) and through the arterial system (one‐third). The sequence of blood entering the liver is first arterial (10 to 40 seconds), portal (40 to 120 seconds), and then late venous (greater than 120 seconds). This vascular discrimination (similar to the one obtained by contrast CT or MRI) allows the collection of information regarding the circulatory system of a tumour (types of feeding vessels, tumour circulatory volume). The presence of arteriovenous communications is characteristic for the neoplastic circulation and in CEUS is expressed by the washout process. This phenomenon begins at the end of the arterial phase or during the venous phase (or both), it is persistent, and is characteristic for neoplastic processes in 90% of cases. Studies that correlate the washout speed of the tumour with its aggressiveness exist, attributing CEUS a prognostic value (Jang 2007; Liu 2007; Bhayana 2010; Boozari 2011).

Type of contrast agents

The first‐generation contrast media, such as Levovist (Bayer Schering Pharma, Berlin, Germany), consisted of air (99.9%) and palmic acid (0.1%) contained within a shell of galactose microparticles. They were found to be unstable, and later, around 2004, they were replaced by second‐generation contrast media such as SonoVue (Bracco, Milano, Italy), Definity (marketed in North America as Luminity by Lantheus Medical Imaging, North Billerica, MA, USA), Optison (GE Healthcare, Princeton, NJ, USA), and Sonazoid (GE Healthcare, Oslo, Norway). SonoVue consists of sulphur hexafluoride contained within a phospholipid shell. Sulphur hexafluoride is an inert molecule that does not interact with any other in the body. Definity consists of octafluoropropane gas contained within a lipid shell, and Optison consists of octafluoropropane within an albumin shell. Both have been approved only for cardiac applications. Blood‐pool agents (e.g. Sonovue) and blood‐pool/Kupffer cell agent (e.g. Sonazoid (perfluorobutane), that also provides the Kupffer phase, which usually starts from 10 to 15 minutes to 120 minutes after Sonazoid administration) will be analysed separately. The 2017 version of LI‐RADS CEUS criteria apply only to blood‐pool agents and not to the blood‐pool/Kupffer cell agents. Therefore, no criteria exist for features in the Kupffer phase of the examination (LI‐RADS 2017).

The characteristic feature of a blood‐pool/Kupffer cell agent (e.g. Sonazoid) is the accumulation in the reticuloendothelial system such as in the liver and spleen. This unique feature of Sonazoid allows the visualisation and interpretation of liver parenchyma during the postvascular phase (i.e. Kupffer phase). The imaging in the Kupffer phase is stable from 10 to 120 seconds after contrast injection, and tolerable for multiple scanning. Malignant hepatic tumours contain few or no Kupffer cells, which can be seen as a perfusion defect in the Kupffer phase.

The accumulation of microbubbles begins immediately after its arrival at liver parenchyma, therefore the phase following the portal venous phase could be determined as the vasculo‐Kupffer phase (1 to 10 minutes), which is presented by a time–intensity curve. Thus, the 'pure portal venous phase' may be very short in Sonazoid‐performed CEUS, determined as the phase from 45 to 60 seconds after injection.

Positivity criteria

Positivity criteria for HCC are based on arterial hyperenhancement and subsequent washout appearance.

On CEUS examination using blood‐pool agents (e.g. Sonovue), the aspect of the HCC is typical and is characterised by accelerated uptake during the arterial phase (hyperenhancement), contrast washout during the portal venous phase, and a hypoechoic appearance in the delayed phase. The washout speed is conditioned by the degree of cellular differentiation of the tumour: the lower the differentiation, the faster the washout (Bhayana 2010; Boozari 2011).

The typical hallmarks for HCC at CEUS differ slightly to those of CT/MRI; for CEUS, hallmarks are arterial hyperenhancement followed by late (more than 60 seconds) washout of a mild degree (Vogel 2018; Vogel 2019). This definition improves the capacity of CEUS to identify malignant lesions such as intrahepatic cholangiocarcinoma (ICC), which are often not identified as definitively malignant by CT and MRI using conventional vascular criteria. This new CEUS criteria for HCC has already been adopted by the Italian Association for the Study of the Liver (AISF) and by the American Association for the Study of the Liver (AASLD) (AISF 2013; Kim 2017; EASL 2018).

Differential diagnosis between ICC and HCC in people with chronic liver disease or liver cirrhosis is a controversial issue. AASLD guidelines from 2011 removed CEUS from the diagnostic procedure for HCC due to the possibility of false‐positive diagnosis of HCC in people with ICC (Bruix 2011). The decision by the AASLD was based on an article stating that 47.6% of ICC showed homogeneous intense enhancement in the arterial phase and washout in the delayed phase on CEUS; findings that were not distinguishable from HCC (Vilana 2010). However, further studies have shown that the enhancement pattern is somewhat different between the two tumours because HCC is more likely to appear as homogeneous or heterogeneous hyperenhancement, whereas ICC often presents with peripheral rim‐like enhancement or heterogeneous hypoenhancement in the arterial phase (Chen 2010). In the quantitative analysis with the time‐intensity curve, ICC showed a more rapid and marked washout than HCC, although there was significant overlap between the two (Kong 2014). Regarding the size of a suspected liver lesion, ICC smaller than 3 cm is more likely to show homogeneous hyperenhancement in the arterial phase with delayed washout, a finding also typical of HCC (Chen 2010). Therefore, careful interpretation of CEUS is needed in smaller nodules developing in the setting of chronic hepatitis or cirrhosis (or both).

Clinical pathway

CEUS is a technique developed in Asia and Europe where its use is more widespread than in northern America. The role of CEUS in the diagnostic pathway for the non‐invasive diagnosis of HCC is not well defined, and recommendations concerning its use vary according to different clinical guidelines. CEUS is used to further assess and characterise focal liver lesions detected with ultrasound, suspected for HCC either in surveillance programmes or in hospital settings. Based on the CEUS findings, if the lesion has no clear features of HCC, unnecessary further examinations will be reduced. However, if the lesion has a malignant potential, further work‐up (CT or MRI) is warranted. In the case of false‐positive results, patients have to undergo needless CT or MRI; false‐negative results have more severe consequences as HCCs go undetected, especially early‐stage HCCs which are eligible for curative treatment.

CEUS is also used in the case of non‐diagnostic results of CT or MRI. Further testing with liver biopsy is performed only in the case of non‐diagnostic results. In this case, false‐positive results are associated with surgical or medical treatment with a wrong indication, whereas false‐negative results imply the missed detection of potentially curable early‐stage HCC.

These two possible diagnostic pathways, illustrated in Figure 1 and Figure 2, are accepted and recommended by the Asian Pacific Association for the Study of the Liver (APASL) and by the European Association for the Study of Liver Disease (EASL) guidelines (Omata 2017; EASL 2018). On the contrary, the AASLD does not recommend the use of CEUS and claims the need of further studies (Heimbach 2018).

Figure 1.

Role of contras enhanced ultrasound (CEUS) as an add‐on test after ultrasound (US) and before computed tomography (CT) or magnetic resonance imaging (MRI), or both.

Figure 2.

Role of contrast enhanced ultrasound (CEUS) as an add on test after computed tomography (CT) or magnetic resonance imaging (MRI) (or both).

Prior test(s)

CEUS is performed after clinical assessment and abdominal ultrasound with the detection of focal lesion suspected for HCC. For surveillance purposes, non‐contrast abdominal ultrasound is recommended as a first‐line imaging modality in people at risk for detection of HCC (Omata 2017; EASL 2018; Heimbach 2018). It is also used as a diagnostic tool in people with clinical suspicion of HCC for detecting liver lesions. In contrast, in some cases of liver nodules detected with ultrasound, CT, MRI, or a combination of these cannot be diagnostic and CEUS can be used prior to histology. Alpha‐fetoprotein (AFP) can also be used prior to CEUS to assess the malignancy of a focal liver lesion.

Role of index test(s)

CEUS can be used as an add‐on test after clinical assessment and ultrasound, and before further complex and expensive imaging techniques (CT and MRI). CEUS allows further characterisation of focal liver lesions detected with ultrasound, suspected for HCC either in surveillance programmes or in hospital settings in people with clinical suspicion. Based on the CEUS findings, if the lesion has no clear features of HCC, unnecessary further examinations will be reduced. However, if the lesion has a malignant potential, further work‐up (CT or MRI) is warranted.

The other possible role is an add‐on test after CT or MRI in the case of non‐diagnostic results. Only in the case of non‐diagnostic CEUS examination, liver histology is required.

We will to investigate the diagnostic accuracy of CEUS in both diagnostic pathways.

Alternative test(s)

Contrast‐enhanced multiphasic multidetector CT and contrast‐enhanced MRI have been established as relevant non‐invasive modalities for detection and evaluation of liver lesions (Lee 2012; O'Neill 2015). The ability to detect HCC rests on characterising the enhancement patterns in arterial, portal venous, and subsequent phases relative to the surrounding liver tissue. The differences in blood flow and extracellular volume between HCC and normal liver tissue lead to main radiological hallmarks such as homogeneous (non‐rim like) arterial phase hyperenhancement suggesting tumoural neo‐angiogenesis and subsequent non‐peripheral washout with enhancing capsule in later phases suggesting the presence of arteriovenous communications (Hennedige 2012; Choi 2014; Shah 2014; LI‐RADS 2017). CT is a commonly used modality for diagnosing HCC due to its short acquisition time and high spatial resolution. However, MRI offers several beneficial features such as absence of X‐ray radiation and combination of various sequences (multiphasic T1‐ and T2‐weighted sequences, diffusion‐weighted imaging, and apparent diffusion coefficient) in combination with the use of extracellular or hepatocellular (or both) gadolinium‐based contrast agent (Arif‐Tiwari 2014; Roberts 2018).

Rationale

HCC is currently detected by liver ultrasound in people with normal or high AFP during surveillance programmes in people with chronic liver disease. Following ultrasound, the diagnosis is usually confirmed with high levels of AFP with or without CEUS, CT, or MRI. The latter two imaging modalities are also appropriate for staging.

This systematic review represents the second part of our series of systematic reviews about the diagnostic accuracy of the most commonly used modalities for diagnosing HCC in people with chronic liver disease. One of the reviews will include assessment of the diagnostic accuracy of ultrasound and AFP levels which are used as triage tests in the surveillance of HCC (Colli 2019). The present review will assess the accuracy of CEUS for the diagnosis of HCC either as a triage test before CT or MRI, or as an add‐on test after CT or MRI. In both cases, the index test, ensuring an adequate accuracy, can be useful to reduce further testing. The third part will focus on the assessment of CT as a third‐line imaging modality in characterising focal liver lesions (Nadarević 2019). A fourth review assessing the accuracy of MRI for diagnosing HCC is in progress (Tang 2017). We are planning to produce an overview of the reviews that assess abdominal ultrasound and AFP, CEUS, CT, and MRI for the diagnosis of HCC when the series of reviews are published.

Objectives

To assess the diagnostic accuracy of contrast‐enhanced ultrasound (CEUS) for the diagnosis of HCC of any size and at any stage in people with chronic advanced liver disease.

Secondary objectives

To assess the diagnostic accuracy of CEUS for the diagnosis of resectable HCC in people with chronic advanced liver disease. The definition of resectable HCC is a neoplasm amenable to surgical radical resection according to the Milan criteria and the current guidelines, that is a single lesion with a maximum diameter of less than 5 cm or fewer than three lesions with a maximum diameter of 3 cm (Mazzaferro 1996; Omata 2017; EASL 2018; Heimbach 2018).

To identify potential sources of heterogeneity, we will investigate the effects of the following: study date; inclusion of participants without cirrhosis; study location; different role of CEUS in the diagnostic clinical pathway; different HCC stage; different liver cirrhosis aetiology; different reference standard; mean HCC diameter; prevalence of the target condition; and type of contrast media.

Methods

Criteria for considering studies for this review

Types of studies

We will include studies that, irrespective of publication status and language, have evaluated the diagnostic accuracy of CEUS for the diagnosis of HCC in people with chronic liver disease. In these studies, all participants should have undergone one of the acceptable reference standards (see Reference standards).

We will consider studies of cross‐sectional design including participants with clinical or sonographical suspicion of HCC. We will exclude studies of case‐control design that compared people with known HCC to matched controls and are considered at high risk of bias inflating accuracy estimates (Colli 2014). We will exclude studies that analysed data only per lesion, rather than per participant, unless we received participant data from study authors.

Participants

Participants will include adults of any age and sex with advanced chronic liver disease, irrespective of aetiology, severity of disease, and duration of illness, with suspect of having HCC on the basis of the results of ultrasound, CT, or MRI. The review will focus on diagnostic questions related to people with a first diagnosis of HCC.

People with previous diagnosis and treatment of HCC make up a distinct group, for which the diagnosis or natural history of HCC were modified. These people are not the focus of this review, and, therefore, we will exclude studies that included such participants unless they represent less than 5% of all the included participants, or if investigators had presented data in such a way as to allow this group of participants to be isolated from the remaining included participants.

Index tests

Contrast‐enhanced ultrasound (CEUS) for detection of HCC in people with advanced chronic liver disease.

CEUS is considered definitely positive with the following features.

• For blood‐pool agents (e.g. Sonovue) when hyperenhancement in arterial phase and late washout (60 seconds or greater) features are detected (LI‐RADS 2017). Hyperenhancement should not be rim‐like or peripheral discontinuous.

• For blood‐pool/Kupffer when abundant tumour vessels appearing as basket‐like or irregular branched shapes from the periphery to the centre of the lesion, and dense tumour staining in the early vascular phase, and fast washout in the late vascular phase, and complete Kupffer defect are detected (Kudo 2008).

The results are dichotomous: positive if all the criteria are present, non‐diagnostic/negative if at least one criterion is absent.

Target conditions

• Hepatocellular carcinoma (HCC) of any size and at any stage.

• Resectable HCC (see above).

Reference standards

We will use different reference standards according to the role of the index test that the included studies aimed to assess.

For studies assessing the role of CEUS as an add‐on test after ultrasound and before CT or MRI, we will accept one of the following, as a reference standard for the diagnosis of HCC:

• typical characteristics on cross‐sectional multiphasic contrast CT or MRI with a follow‐up period of at least six months, to allow the confirmation of an initial negative result of CT or MRI;

• the pathology of the explanted liver in case of transplantation;

• the histology of resected focal liver lesion(s), or the histology of resected or biopsied focal liver lesion(s) and a follow‐up period of at least six months to exclude the presence of focal lesions not detected by the index test.

For studies assessing the role of CEUS as an add‐on test after non‐diagnostic CT or MRI results, we will accept the following:

• the pathology of the explanted liver in case of transplantation;

• the histology of resected focal liver lesion(s), or the histology of resected or biopsied focal liver lesion(s) and a follow‐up period of at least six months to exclude the presence of focal lesions not detected by the index test.

All the accepted reference standards are currently used. The pathology of the explanted liver can be regarded as perfectly accurate, but it is obviously possible only when all the included patients undergo liver transplantation; therefore, the setting does not correspond to the clinical question as only people with advanced and decompensated liver disease can be candidates for orthotopic liver transplantation. CT and MRI are not perfectly accurate as the pathology of explanted liver, but their accuracy is considered sufficient to guide further clinical decisions; moreover, an appropriate follow‐up is required to confirm a negative result. The histology of resected specimen or of lesion biopsy may have false‐negative results and requires follow‐up to exclude the presence of HCC undetected by CEUS. In order to minimise verification bias, we will include only studies in which all participants underwent one of the acceptable reference standards; however, using histology, a differential verification is unavoidable and an appropriate follow‐up is required to confirm their negative results. We do not plan any hierarchy of the reference standards in the case of multiple presentation in the same study and intend to evaluate different reference standards as possible sources of heterogeneity.

Search methods for identification of studies

Electronic searches

We will search the Cochrane Hepato‐Biliary Group (CHBG) Controlled Trials Register and the Cochrane Hepato‐Biliary Group Diagnostic Test of Accuracy Studies Register (both are maintained and searched internally by the CHBG Information Specialist via the Cochrane Register of Studies Web), the Cochrane Library, MEDLINE Ovid, Embase Ovid, LILACS (Bireme), Science Citation Index – Expanded (Web of Science), and Conference Proceedings Citation Index – Science (Web of Science) (Royle 2003).

We will apply no language or document type restrictions.

Searching other resources

We will identify additional references by manually searching articles retrieved from digital databases and relevant review articles. We will seek information on unpublished studies by contacting experts in the field. In addition, we will handsearch abstract books from meetings of the AASLD, EASL, and APASL held over the 10 years prior to the search. We will search for other types of grey literature in the System for Information on Grey Literature in Europe 'OpenGrey' (www.opengrey.eu/).

Data collection and analysis

We will follow available guidelines as provided in the Cochrane Handbook for Diagnostic Test of Accuracy Reviews (DTA Handbook 2013).

Selection of studies

We will use Covidence to manage the selection of studies (Covidence 2019). Two review authors (VG and TN) will independently scrutinise titles and abstracts identified by electronic literature searching to identify potentially eligible studies. We will select any citation, identified by either of the two review authors, as potentially eligible for full‐text review. The same review authors will independently assess full‐text papers for study eligibility, using predefined inclusion and exclusion criteria. We will resolve any discrepancies by discussion. We will record all studies after full‐text assessment and their reasons for exclusion in the 'Characteristics of excluded studies' table and illustrate the study selection process using a PRISMA diagram (Moher 2009).

Where studies have multiple publications, we will collate the reports of the same study so that each study, rather than each report, is the unit of interest for the review, and such studies have a single identifier with multiple references.

Data extraction and management

Two review authors (MF and TN), working as a pair, will complete a prepiloted data extraction form for each included study. Each review author will independently extract study data. In cases of discordance, we will reach consensus through discussion.

We will retrieve the following data and report them in the 'Characteristics of included studies' table:

• general information: title, journal, year, publication type, and study design (prospective versus retrospective);

• sample size: number of participants meeting the criteria and total number of participants assessed;

• baseline characteristics: baseline diagnosis, age, sex, race, presence of cirrhosis, and mean diameter of HCC;

• index test with predefined positivity criteria and its role in the clinical pathway;

• reference standard tests;

• numbers of true‐positive, true‐negative, false‐positive, and false‐negative findings. We will extract these data for the two target conditions (HCC of any size, stage and resectable HCC).

We will summarise the data from each study in 2 × 2 tables (true positive, false positive, false negative, true negative), according to the index tests considered, and we will enter the data into Review Manager 5 software (Review Manager 2014).

Missing data

We will contact primary authors by e‐mail to ask for missing data which are needed to complete the 2 × 2 tables. If we receive no reply, we will send a second e‐mail after two weeks. If no reply is received, we will exclude the study in question.

Assessment of methodological quality

Two review authors (MF and TN) will independently assess the risk of bias of included studies and applicability of their results using QUADAS‐2 (revised tool for quality assessment of diagnostic accuracy studies) (Whiting 2011). In cases of discordance, we will reach a consensus through discussion. We will address aspects of study quality involving the participant spectrum, index tests, target conditions, reference standards, and flow and timing. We added a specific signalling question in QUADAS‐2 domain 'Flow and timing': "Were participants with non‐evaluable result of the index test included and analysed according to intention‐to‐diagnose principle (non‐evaluable results considered as false)?". We will classify a study as having risk of bias if at least one of the domains of QUADAS‐2 is at high‐risk of bias.

Statistical analysis and data synthesis

We will carry out statistical analyses according to recommendations provided in the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy (DTA Handbook 2013). We will design 2 × 2 tables (true positive, false positive, false negative, true negative) for each primary study.

First, we will perform a graphical descriptive analysis of the included studies. We will present forest plots (sensitivity and specificity separately, with their 95% confidence intervals (CIs)), and we will provide a graphical representation of studies in the receiver operating characteristic space (sensitivity against 1 – specificity). Second, we will perform a meta‐analysis using the bivariate model and will provide estimates of summary sensitivity and specificity. We will use the pooled estimates obtained from the fitted models to calculate summary estimates of positive (LR+) and negative (LR–) likelihood ratios.

We anticipate that ultrasound visualisation and hence contrast disposition can often be suboptimal due to patient characteristics; therefore, lack of reporting or excluding those cases from analyses could overestimate the accuracy of CEUS. The clinical consequence of non‐evaluable results is the need of further testing (CT, MRI, or biopsy). Including non‐evaluable results and considering them as false positives and false negatives seems to summarise the diagnostic accuracy and true clinical potential most adequately. Hence, in case of non‐evaluable index test results, we will analyse data according to the intention‐to‐diagnose (ITD) principle (Schuetz 2012), also described as the worst‐case scenario (Cohen 2016). Participants with indeterminate index test results will be classified as false positive if they had a negative reference standard or a false negative for participants with a positive reference standard. If data for the ITD analyses are not retrievable from the text, we will contact study authors with provided e‐mail addresses. In case of no reply, we will include the study in the analyses with data retrievable from the published manuscript and considered it at high risk of bias (see 'Missing data').

We will perform all statistical analyses using SAS statistical software, release 9.4 (SAS Institute Inc., Cary, NC, USA) and macro METADAS (DTA Handbook 2013).

Investigations of heterogeneity

We will investigate the effects of the following potential sources of heterogeneity:

• study date: studies published in 2004 or earlier compared to studies published after 2004 due to advancements in technology study date (categorical covariate);

• inclusion of participants without cirrhosis: studies including 10% or greater participants without cirrhosis compared to studies including less than 10% participants without cirrhosis (categorical covariate);

• study location (population differences): studies conducted in Americas compared to Europe compared to Asia (categorical covariate);

• different role of CEUS in the diagnostic clinical pathway: studies using CEUS after ultrasound compared to studies using CEUS after CT and MRI (categorical covariate);

• different HCC stage: studies with 20% or greater of resectable HCC compared to studies with less than 20% of resectable HCC (categorical covariate);

• different liver cirrhosis aetiology: hepatitis C or hepatitis B virus‐associated cirrhosis compared to all other aetiologies (categorical covariate);

• different reference standard: studies using the pathology of the explanted liver compared to liver biopsy compared to other reference standards (categorical covariate);

• mean diameter of the cancer (continuous covariate);

• prevalence of the target condition (continuous covariate);

• type of contrast media: blood‐pool versus bloodpool/Kupffer cell (categorical covariate).

We chose the above listed variables for the following reasons. Due to advancements in technology and change in diagnostic criteria, we considered the date of study publication. The proportion of participants without cirrhosis is relevant because HCC in the absence of cirrhosis has different CT characteristics, prognosis, and treatment. In epidemiological studies, this proportion is usually less than 10% (Forner 2018). There are differences in epidemiology and clinical and radiological characteristics of HCC in Asia when compared to western countries. The HCC prevalence in included studies can change accordingly to selection and epidemiology. Assessing the accuracy of CEUS in a different role of the diagnostic pathway implies different characteristics of participants, different reference standards, and expected different results. The proportion of resectable HCC found in the studies reflects different epidemiology and participants selection. The clinical and radiological characteristics of HCC varies according to the aetiology of the underlying liver disease, mainly in case of chronic hepatitis C virus or hepatitis B virus infection compared to other aetiologies. The accuracy of CEUS may vary according to the different reference standard, diameter of the neoplastic lesion, the type of contrast used, and the definition of positivity criteria. We will estimate effects by adding covariates to the bivariate model. We will assess the statistical significance of the covariate effect using the log‐likelihood ratio test for comparison of models with and without the covariate term. We will consider P values less than 0.05 as two‐sided and statistically significant.

Sensitivity analyses

We will assess effects of risk of bias of included studies on diagnostic accuracy by performing a sensitivity analysis from which we will exclude studies with the following characteristics:

• studies classified as at high risk of bias, that is, studies having high risk of bias in at least one of the domains of QUADAS‐2 (Appendix 2). In addition, we have defined the following signalling questions as most relevant, and will assess them in separate sensitivity analyses, excluding studies with 'No' or 'Unclear' answers:

• • “Were the positivity criteria defined?”

  • "Were the reference standard results interpreted without the knowledge of the results of the index test?"

  • "Were participants with non‐evaluable result of the index test included and analysed according to the ITD principle (non‐evaluable results considered as false)?"

We will conduct a sensitivity analysis in excluding studies published only in abstract or letter form.

Assessment of reporting bias

We will not test for publication bias due to the lack of validated methods for diagnostic test accuracy reviews.

'Summary of findings' table

We will prepare a Summary of findings table to present the main results and key information regarding the certainty of evidence, using the GRADE assessment (GRADEpro GDT).

We will apply the GRADE judgements for the GRADE domains as following:

• risk of bias: we will use QUADAS‐2 to assess risk of bias;

• indirectness: we will use QUADAS‐2 for concerns of applicability and look for important differences between the populations studied (e.g. the spectrum of disease), the setting, and the index test;

• inconsistency: we will carry out prespecified analyses to investigate potential sources of heterogeneity and we will downgrade the evidence when we cannot explain inconsistency in the accuracy estimates;

• imprecision: we will look at the CIs of sensitivity and specificity estimates and at the unexplained heterogeneity of the results;

• publication bias: we will not evaluate publication bias as such validated methods for diagnostic test accuracy reviews are lacking.

We will justify all decisions to downgrade the quality of studies using footnotes and we will make comments to aid reader's understanding of the review where necessary.

Appendices

Appendix 1. Search strategies

Database	Time span	Search strategy
Cochrane Hepato‐Biliary Group Controlled Trials Register	Date will be given at review stage.	(ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*) AND (((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC) AND ((advanc* or chronic) and (liver* or hepat*))
Cochrane Hepato‐Biliary Group Diagnostic Test of Accuracy Studies Register	Date will be given at review stage.	(ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*) AND (((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC) AND ((advanc* or chronic) and (liver* or hepat*))
The Cochrane Library	Latest issue	#1 MeSH descriptor: [Ultrasonography] explode all trees #2 (ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*) #3 #1 or #2 #4 MeSH descriptor: [Carcinoma, Hepatocellular] explode all trees #5 MeSH descriptor: [Liver Neoplasms] explode all trees #6 (((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC) #7 #4 or #5 or #6 #8 ((advanc* or chronic) and (liver* or hepat*)) #9 #3 and #7 and #8
MEDLINE Ovid	1946 to the date of search	1. exp Ultrasonography/ 2. (ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*).mp. [mp=title, abstract, original title, name of substance word, subject heading word, floating sub‐heading word, keyword heading word, protocol supplementary concept word, rare disease supplementary concept word, unique identifier, synonyms] 3. 1 or 2 4. exp Carcinoma, Hepatocellular/ 5. exp Liver Neoplasms/ 6. (((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC).mp. [mp=title, abstract, original title, name of substance word, subject heading word, floating sub‐heading word, keyword heading word, protocol supplementary concept word, rare disease supplementary concept word, unique identifier, synonyms] 7. 4 or 5 or 6 8. ((advanc* or chronic) and (liver* or hepat*)).mp. [mp=title, abstract, original title, name of substance word, subject heading word, floating sub‐heading word, keyword heading word, protocol supplementary concept word, rare disease supplementary concept word, unique identifier, synonyms] 9. 3 and 7 and 8
Embase Ovid	1974 to the date of search	1. exp echography/ 2. exp ultrasound/ 3. (ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*).mp. [mp=title, abstract, heading word, drug trade name, original title, device manufacturer, drug manufacturer, device trade name, keyword, floating subheading word, candidate term word] 4. 1 or 2 or 3 5. exp liver cell carcinoma/ 6. exp liver tumor/ 7. (((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC).mp. [mp=title, abstract, heading word, drug trade name, original title, device manufacturer, drug manufacturer, device trade name, keyword, floating subheading word, candidate term word] 8. 5 or 6 or 7 9. ((advanc* or chronic) and (liver* or hepat*)).mp. [mp=title, abstract, heading word, drug trade name, original title, device manufacturer, drug manufacturer, device trade name, keyword, floating subheading word, candidate term word] 10. 4 and 8 and 9
LILACS (Bireme)	1982 to the date of search	((advanc$ or chronic) and (liver$ or hepat$)) [Words] and (((liver or hepato$) and (carcinom$ or cancer$ or neoplasm$ or malign$ or tumo$)) or HCC) [Words] and (ultrasound or ultrasonogra$ or US or CEUS or sonogra$ or echogra$ or echotomogra$) [Words]
Science Citation Index Expanded (Web of Science)	1900 to the date of search	#4 #3 AND #2 AND #1 #3 TS=((advanc* or chronic) and (liver* or hepat*)) #2 TS=(((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC) #1 TS=(ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*)
Conference Proceedings Citation Index – Science (Web of Science)	1990 to the date of search	#4 #3 AND #2 AND #1 #3 TS=((advanc* or chronic) and (liver* or hepat*)) #2 TS=(((liver or hepato*) and (carcinom* or cancer* or neoplasm* or malign* or tumo*)) or HCC) #1 TS=(ultrasound or ultrasonogra* or US or CEUS or sonogra* or echogra* or echotomogra*)

Appendix 2. QUADAS‐2

Domain	1. Participant selection	2. Index test	3. Reference standard	4. Flow and timing
Signalling questions and criteria	Q1: "Was a consecutive or random sample of participants enrolled?" Yes – if the study reports on a consecutive or a random selection of participants. No – if the study reports on another form of selection of participants. Unclear – if the study does not report on how the participants were enrolled. Q2: "Did the study avoid inappropriate exclusions?" Yes – if definitions of exclusion criteria are appropriate (i.e. previous surgery or treatment for HCC; people with cholangiocarcinoma) and all exclusions are reported. No – if exclusion criteria are inappropriate and exclusions are not reported. Unclear – if the study does not report causes of exclusions.	Q1: "Were the index test results interpreted without knowledge of the results of the reference standard?" Yes – if the study reports that the results of the index test were interpreted without the knowledge of the results of the reference standard. No – if the study reports that results of the index test were interpreted with the results of the reference standard. Unclear – if the study does not report information about blinding of the results of the index test and reference standard. Q2: "Were positivity criteria clearly defined?" Yes – if the study clearly reports positivity criteria (i.e. for blood‐pool agents when hyperenhancement in arterial phase and a late washout (≥ 60 seconds) features are detected. For blood‐pool/Kupffer cell agent when abundant tumour vessels appearing as basket‐like or irregular branched shapes from the periphery to the centre of the lesion, and dense tumour staining in the early vascular phase and fast washout in the late vascular phase, and complete Kupffer defect are detected). No – if the study does not report the positivity criteria.	Q1: "Is the reference standard likely to correctly classify the target condition?" Yes – if the reference standard correctly defines the presence/absence of HCC such as pathology of explanted liver in a transplant cohort). No – if other reference tests than pathology of explanted liver were used, such histology of resected specimen or of focal lesion biopsy. Q2: "Were the reference standard results interpreted without the knowledge of the results of the index test?" Yes – if the study reports that the results of the reference standard were interpreted without the knowledge of the results of the index test. No – if the study reports that the results of the reference standard were interpreted with the knowledge of the results of the index test. Unclear – if the study does not report information about blinding of the results of the reference standard and the index test.	Q1: "Was there an appropriate interval between the index test and the reference standard?" Yes – if the interval between the index test and the reference standard was less than 3 months. No – if the interval was equal or longer than 3 months. Unclear – if the study does not report the interval between the index test and the reference standard. Q2: "Did all participants receive the same reference standard?" Yes – if the study has only 1 reference standard for all the participants. No – if the study has more > 1 reference standard. Unclear – if the study information regarding the use of reference standard are unclear. Q3: "Were all participants included in the analysis and analysed according to intention‐to‐diagnose principle (non‐evaluable results considered as false)?" Yes – if all enrolled participants were included in the analysis. No – if any participant was excluded from the analysis for any reason. Unclear – if the exclusion of participants from the analysis is unclear. Q4. "Were participants with non‐evaluable result of the index test included and analysed according to intention‐to‐diagnose principle (non‐evaluable results considered as false)?" Yes – if participants with non‐evaluable results were included and analysed according to intention to diagnose principle. No – If participants with non‐evaluable results were not included and analysed according to intention‐to‐diagnose principle.
Risk of bias	Could the selection of participants have introduced bias? If we answer 'yes' to all signalling questions, then we will judge the risk of bias as 'low'. If we answer 'no' to at ≥ 1 of the signalling questions, then we will judge the risk of bias as 'high'. If we answer 'unclear' to all signalling questions, then we will judge the risk of bias as 'unclear'. If we answer 'unclear' to ≥ 1 of the signalling questions and to the remaining our answer is 'yes', then we will judge the risk of bias as 'unclear'.	Could the conduct or interpretation of the index test have introduced bias? If we answer 'yes' to all signalling questions, then we will judge the risk of bias as 'low'. If we answer 'no' to ≥ 1 of the signalling questions, then we will judge the risk of bias as 'high'. If we answer 'unclear' to all signalling questions, then we will judge the risk of bias as 'unclear'. If we answer 'unclear' to ≥ 1 of the signalling questions and to the remaining our answer is 'yes', then we will judge the risk of bias as 'unclear'.	Could the reference standard, its conduct, or its interpretation have introduced bias? If we answer 'yes' to all signalling questions, then we will judge the risk of bias as 'low'. If we answer 'no' to ≥ 1 of the signalling questions, then we will judge the risk of bias as 'high'. If we answer 'unclear' to all signalling questions, then we will judge the risk of bias as 'unclear'. If we answer 'unclear' to ≥ 1 of the signalling questions and to the remaining our answer is 'yes', then we will judge the risk of bias as 'unclear'.	Could the participant flow have introduced bias? If we answer 'yes' to all signalling questions, then we will judge the risk of bias as 'low'. If we answer 'no' to ≥ 1 of the signalling questions, then we will judge the risk of bias as 'high'. If we answer 'unclear' to all signalling questions, then we will judge the risk of bias as 'unclear'. If we answer 'unclear' to ≥ 1 of the signalling questions and to the remaining our answer is 'yes', then we will judge the risk of bias as 'unclear'.
Concerns about applicability	Are there concerns that included participants and setting do not match the review question? Low concern: the participants included in the review represent the participants in whom the test is used in clinical practice (i.e. second‐line imaging modality in people with suspected liver lesion). High concern: the participants included in the review differ from the participants in whom the test is used in clinical practice.	Are there concerns that the index test, its conduct, or interpretation differ from the review question? Low concern: the index test, its conduct, or its interpretation does not differ from the way it is used in clinical practice. High concern: the index test, its conduct, or its interpretation differs from the way it is used in clinical practice.	Are there concerns that the target condition as defined by the reference standard does not match the question? High concern: the definition of the target condition as defined by the reference standard does not match the question (i.e. pathology of the explanted liver is feasible only in the case of liver transplant; the natural history and prognosis of HCC detected in explanted liver might be different). Low concern: the definition of the target condition as defined by the reference standard does match the question, e.g. CT scan or MRI for all included participants.	—
CT: computer tomography; HCC: hepatocellular carcinoma; MRI: magnetic resonance imaging.

Contributions of authors

MF: wrote the protocol, will perform searches for references, evaluate references for obtaining the full reports, evaluate studies for inclusion, extract data from studies, assess the risk of bias, and write the final review. TN: wrote the protocol, will perform searches for references, evaluate references for obtaining the full reports, evaluate studies for inclusion, extract data from studies, assess the risk of bias, and write the final review. VG: commented on the protocol, and will critically comment on the final review. AC: co‐ordinated protocol design, and will design the final review. DM: commented on the protocol, and will critically comment on the final review. DŠ: critically commented on the protocol, will act as arbiter if review authors cannot reach a consensus, and will critically comment on the final review. GC: wrote the protocol, and will critically comment on the final review.

Sources of support

Internal sources

• No sources of support supplied

External sources

• None, Other.

  None

Declarations of interest

MF: none. TN: none. VG: none. AC: none. DM: none. DŠ: none. GC: none.

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