Combined hepatocellular-cholangiocarcinoma (CHC) is a rare subtype of primary liver cancer (PLC) presenting clinical and pathological distinctions of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) simultaneously (1). In 1949, Allen and Lisa (2) categorized CHC as type A (double type), type B (combined type), and type C (mixed type). However, the recent 2019 WHO classification excised other subcategorizations and simplified the definition of CHC as an entity containing an intimate mixture of HCC and intrahepatic cholangiocarcinoma (ICC) components, which was the previously reported Allen C type (3). Due to the changing definitions and nomenclature systems, its reported incidence varied from 0.4% to 14.2% among PLCs (4). It is well known that the most frequent PLCs are HCC and ICC (5).

Extensive studies suggested that CHC tended to occur at chronic liver damage and subsequent cirrhosis, which were the known risk factors of HCC and ICC (6). However, treatment strategies differed substantially among the three diseases. Major hepatic resection with lymph node dissection was the recommended treatment for CHC (7). Trans-arterial chemoembolization (TACE) and locoregional therapies, which can be served as the curative therapy for certain HCCs and ICCs, did not show comparable outcomes in the treatment of CHC (8–11). As for systemic therapy, many chemotherapeutic agents and molecular-targeted drugs for HCC have been developed and drawn encouraging results (sorafenib, nivolumab, etc.) (12, 13). And ICC was reported to harbor the FGFR2 fusions or IDH1/2 mutations, for which infigratinib was developed and now under phase II clinical trial (14). However, systemic therapy regimens for unresectable CHC remain controversial because of its dual nature (15, 16). Liver transplantation had demonstrated inferior survival benefits and higher relapse rates in CHC compared with HCC, which should be avoided in CHC patients so as to allocate liver sources for more appropriate candidates (15, 17). The prognosis of CHC was reported as either intermediate between HCC and ICC or worse than that of both malignancies (18–20). Therefore, imaging differentiation of CHC from HCC and ICC is crucial in terms of proper treatment decision and better survival outcome.

B-mode ultrasound (BMUS) now serves as the first-line imaging modality for tumor detection or follow-up in high-risk patients (21). The development of contrast-enhanced ultrasound (CEUS) enables real-time visualization of the microcirculation within nodules, thus providing more useful diagnostic information. However, owing to the histological diversity and complex existing forms of tumor components, CHC can display either an HCC-like pattern or an ICC-like pattern on CEUS, which makes it difficult to distinguish CHC from HCC and ICC (22). The diagnostic criteria of Liver Imaging Reporting and Data System (LI-RADS) was launched for standardizing the interpretation of liver imaging and classification of hepatic neoplasms (23). However, imaging misclassification has been reported in approximately half of CHC lesions by using the CEUS LI-RADS category (24, 25). The concomitant increase of alpha fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9) seems to be of moderate diagnostic value, but the sensitivity (17.8%) was too low (26). Besides, the combination of tumor markers and CEUS findings (i.e., elevated CA19-9 with imaging features of HCC pattern) only reaches moderate success to diagnose CHC (73.3%–76.9% accuracy) (27, 28). Thus, imaging techniques alone or with the help of LI-RADS and tumor markers cannot provide a reliable preoperative diagnosis of CHC and remain inadequate in the instruction of management.

Nomogram is a feasible and relatively objective tool to predict the individual probability of a clinical event, which has been established and validated to be effective in a substantial proportion of cancer types. Here we developed a nomogram based on clinical indicators, BMUS characteristics, and CEUS features that are selected by least absolute shrinkage and selection operator (LASSO) regression analysis, expecting to preoperatively differentiate CHC from HCC and ICC as well as facilitate clinical decision-making.

CHC is the third most common PLC with the incidence only second to HCC and ICC. Preoperative identification of CHC and non-CHC PLC on imaging is necessary with regard to the proper treatment decision for better prognosis. However, as a biphasic tumor formed by a mixture of hepatocellular and biliary components, the imaging findings of CHC have always been elusive.

A nomogram is able to quantify the risk of a clinical event through an intuitive graph of a statistical predictive model, which has been widely developed for the auxiliary diagnosis and prognostic prediction of malignancy (30). Wang et al. (31) established a nomogram based on clinical indicators to differentiate CHC from ICC, which achieved a good discriminating capability (C-index of 0.796). To make preoperative identification of CHC and fully exploit the advantage of BMUS and CEUS, we constructed a nomogram incorporating clinical indexes along with imaging characteristics of BMUS and CEUS. The nomogram integrated five parameters selected by LASSO regression, including irregular shape and obscure boundary on BMUS, the enhanced pattern on CEUS, and the serologic test of elevated AFP level as well as SPE α1 level. The utilization of LASSO regression effectively reduced the impact of multicollinearity and model overfitting (32).

Malignancy is characterized by the presence of irregular shape and ill-defined boundary. Ye et al. (33) found that a higher proportion of ill-defined boundaries was observed in CHCs than that in HCCs. In accordance with their study, indistinct margin and irregular shape presented more frequently in the CHC and ICC lesions than in the HCC nodules. One plausible reason might be that cholangiocarcinoma-containing tumors, whose tumor cell partly arises from cholangiocyte, have the property of infiltrative progression (34). In the present study, homogeneous hyperenhancement was more frequently found in CHC, whereas heterogeneous hyperenhancement was more commonly identified in HCC and ICC. Inconsistently, the studies from Huang et al. (28) and Zhang et al. (26) found that heterogeneous hyperenhancement was more common in CHC and ICC, and homogeneous hyperenhancement was more frequently observed in HCC, which might be due to the high degree of heterogeneity in CHC. It was believed that the CEUS patterns of CHC nodules might be influenced by the relative proportions of the HCC/ICC components within nodules (35). Regardless, the different CEUS patterns of nodules were verified as a predictor in distinguishing different subtypes of PLC, the mechanism of which needs be further investigated. AFP is the widely used tumor marker for PLC, especially for the HCC-containing tumors (36). In our study, the elevation of AFP was more common in the HCC and CHC than in ICC, which was consistent with the study by Zhang et al. (26). They found that elevated AFP was observed in 71.1% of HCC, 55.6% of CHC, and 2.2% of ICC. Reason for such a discrepancy might be that AFP was synthesized by liver cells, whereas ICC mainly originated from the epithelial cells of the intrahepatic bile duct. This finding may help rule out the ICC diagnosis among PLCs. SPE is an inexpensive and useful tool to assess liver function. An increase of the α1 globulins and α2 globulins band was observed in many malignant tumors due to the increase of acute-phase proteins (37). Differences of SPE Alb and SPE α1 level between the CHC group and non-CHC group were observed in our study, and SPE α1was further confirmed as a predictor by LASSO regression to identify CHC and non-CHC lesions. However, the cause of this discrepancy has not yet been determined.

The nomogram we established is an intuitive and easy-to-use tool for the individualized prediction of a CHC diagnosis. It performed well in differentiating CHC from non-CHC nodules, achieving the C-index of 0.8275 in the training set and 0.8530 in the validation set. Good agreement between predictions and observations underlined the reliability and repeatability of this nomogram. The nomogram will help identify the CHC nodules previously misdiagnosed as HCC or ICC and lead to more appropriate treatment plans. For instance, a patient with elevated AFP level (25.4 ng/ml) and SPE α1 level of 2.9% revealed a 45 × 30-mm mass on BMUS with irregular shape and obscure boundary ( Figure 6A ). The nodule displayed rim hyperenhancement in the arterial phase on CEUS ( Figures 6B, C ). On the basis of the nomogram, the possibility of CHC diagnosis was 0.91 ( Figure 3 ). Postoperative pathological examination confirmed a diagnosis of CHC ( Figures 6D–F ). Therefore, curative hepatectomy with lymph node dissection would be the best treatment option for this patient. DCA also found that this CEUS nomogram added more benefit than either the treat-none scheme or the treat-all-patients strategy. Beyond that, the ability of the nomogram in evaluating the personalized probability of CHC diagnosis will facilitate patient selection for prospective clinical trials.

Previously, researchers have attempted to identify the three different types of PLCs by imaging modalities. Wang et al. (38) tried to identify HCC, ICC, and CHC based on their preoperative CT and MRI, through which they found that imaging features of capsular retraction, biliary dilatation, pseudocapsule, rim enhancement, and abnormal perfusion differed significantly between the three malignancies, and CHC was intermediate between HCC and ICC. However, they also pointed out that the enhancement pattern of CHC resembled HCC in most cases. Immediately after, LI-RADS was brought into the attempts of CHC diagnosis. Choi et al. (39) evaluated the images of 194 PLC nodules (53 with CHC, 44 with ICC, and 97 with HCC) with gadoxetic acid-enhanced MRI and classified them according to the LI-RADS; they found that the misdiagnosis of HCC was mainly due to the confusing imaging manifestation of CHC, since 85% of false-positive diagnosis of HCC (LR-5) was CHC. Compared with CT or MRI, ultrasound technology features simple operation and real-time observation, which played an important role in the diagnosis and intervention of liver neoplasms.

The ability of CEUS in making differentiation of CHC from HCC and ICC was also investigated. The study of Sagrini et al. (40) compared the ability of different imaging modalities (CEUS, CT, and MRI) in making diagnosis of CHC, which revealed that CEUS possessed strong diagnostic ability in distinguishing malignant from benign liver lesions while it failed to make a definitive diagnosis of CHC in most cases. On this basis, a combination of CEUS and tumor markers was carried out (simultaneous elevation of AFP and CA19-9 or elevated tumor markers in discordance with the imaging findings). However, this criterion also presented limited diagnostic capacity, with the accuracy of 71.1%–74.4% (22, 26, 28). Recent studies further investigated the value of CEUS LI-RADS combined with tumor markers in making a diagnosis of CHC. Nevertheless, the accuracy (76.9%) showed no significant improvement compared with CEUS only (27). Comparing with the mentioned diagnostic modalities, our nomogram not only incorporated tumor markers and CEUS characteristics but also contained BMUS features and indicator of liver function test, which were the underestimated factors in previous studies, and the diagnostic power was therefore improved.

Ultrasound radiomics was the major research focus currently, which greatly enhanced the imaging diagnostic efficiency for hepatic tumors. Peng et al. (41) extracted radiomics features from the grayscale ultrasound images of 668 PLCs and constructed the radiomics model. Their model displayed a high diagnostic value in identifying HCC, ICC, and CHC, with AUCs of 0.920 (training cohort) and 0.728 (test cohort). Nevertheless, the application of the radiomics models required specific techniques and software, which was not user-friendly (42). On the contrary, our nomogram not only shows high diagnostic effectiveness but also incorporates few predictors, which will simplify the diagnostic process and be more applicable for less experienced physicians. Additionally, all the three imaging features and two clinical indicators are convenient to obtain from preoperative examination, making the nomogram with high clinical practicability.

Despite the good performance, certain limitations existed in the present study. First, as a single-center study, additional multicenter prospective studies are necessary so as to validate the diagnostic ability of the nomogram. Second, the evaluation of imaging characteristics showed operator dependence. Subjectivity cannot be eliminated entirely. Lastly, heterogeneity of the ultrasound images was inevitable in this retrospective study, since the ultrasound examinations were conducted in different machines by different radiologists. Therefore, a prospective study with designated radiologists and specific ultrasound machine is mandatory.

In conclusion, we developed and validated a nomogram based on ultrasonographic features and clinical characteristics to differentiate CHC from HCC and ICC. Our results suggested that the nomogram had promising predictive power and high clinical practicability, which will be helpful in making preoperative diagnosis and selecting appropriate treatments.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the ethics committee of Zhongshan Hospital, Fudan University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

YC made contribution to the study designed, statistical analysis, and article drafting. QL, WZ, YD, JC, and WW contributed to the ultrasound image acquirement. QL and WW critically reviewed the article and made revisions. All authors participated sufficiently in the study and gave approval of the submitted version.

The study was supported by the National Natural Science Foundation of China (Grant no. 82071924); Natural Science Foundation Project of Shanghai (Grant no. 20ZR1452800); Clinical Research Plan of Shanghai Hospital Development Center (Grant no. SHDC2020CR1031B), and Shanghai Municipal Key Clinical Specialty (Grant no. shslczdzk03501).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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