Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver malignancy, with an increasing incidence and mortality worldwide [1–3]. Currently, surgical resection represents the curative treatment option, but surgery is only possible for selected patients. Even with optimal surgery, the 5-year overall survival (OS) rate is 15–40% [4, 5]. The incidence of post-surgical recurrence is 50–60% [3, 4], and recurrence is associated with a poor prognosis. It is thus essential to develop a multidisciplinary strategy for ICC to improve prognosis [6–9].

Recently, new therapies based on the molecular or genetic characteristics of ICC have been developed. In theory, it should be possible to select the appropriate therapy for each patient depending on the molecular or genetic characteristics of their individual tumors. Without surgical resection, however, there is little biological information in ICC, so it is difficult to select treatment before surgery.

In the era of genetic landscape and precision medicine, increasing evidence suggests that genetic mutation profiles allow the evaluation of prognosis of cancer postoperatively [10–14]. ICC has a relatively large number of actionable mutations compared to other gastrointestinal carcinomas. Several studies have demonstrated that KRAS mutation in ICC could affect prognosis [12–14]. Therefore, assessment of KRAS mutation status may contribute to the development of treatment strategies. A surrogate marker for KRAS mutation would provide genomic information without the need for biopsy or surgery. To identify this surrogate marker, it may be helpful to assess the biological effects of KRAS mutation.

Investigators have reported that tumor cells with KRAS mutation exhibit enhanced glucose uptake and glycolysis to survive in severe conditions (i.e., low-glucose and hypoxia) [15–17]. Glucose transporter-1 (GLUT-1) is a major glucose transporter in cholangiocarcinoma, and its expression is correlated with higher malignant potential in ICC [18, 19]. Positron emission tomography (PET) with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), a glucose analog, is a less invasive modality that determines the glucose metabolism potential of tumors by quantifying ¹⁸F-FDG uptake. However, to date, the association between KRAS mutation and ¹⁸F-FDG-PET has not been reported in ICC.

We investigated the presence of KRAS mutation, the expression of GLUT-1, and ¹⁸F-FDG-PET parameters in 50 ICC patients, and examined whether there was an association between ICC prognosis and these factors.

¹⁸F-FDG-PET studies were performed using a PET/computed tomography (CT) scanner (Discovery ST Elite or Discovery IQ; GE Healthcare, Milwaukee, WI, USA). Patients fasted for at least 4 h before undergoing ¹⁸F-FDG-PET. The plasma glucose level was checked before the injection of ¹⁸F-FDG, and there were no patients with blood glucose level > 150 mg/dL in this study. Data acquisition started approximately 60 min after intravenous administration of ¹⁸F-FDG (injected dose: approximately 3.7 MBq/kg body weight). Initially, starting at the level of the upper thigh, low-dose CT scans were obtained with the following parameters: 40–60 mA, 120 kV, 0.6-s tube rotation, and 3.75-mm section thickness. The CT images were acquired during shallow breathing, and scanning included the area from the upper thigh to the skull. Immediately after the CT scans were acquired, PET emission scanning was performed with an acquisition time of 2–3 min per bed position. Whole-body PET images were attenuation-corrected using CT data and reconstructed using a 3D ordered-subsets expectation–maximization algorithm called VUE Point Plus (Discovery ST Elite: 14 subsets, two iterations, a matrix size of 128 × 128, a voxel size of 4.7 × 4.7 × 3.3 mm, and post-filtering at 5-mm full width at half maximum; Discovery IQ: 12 subsets, four iterations, a matrix size of 192 × 192, a voxel size of 3.3 × 3.3 × 3.3 mm, and post-filtering at 5-mm full width at half maximum). For quantitative analysis, at least two board-certified radiologists/nuclear medicine physicians assessed ¹⁸F-FDG accumulation on a workstation (Advantage Workstation 4.6; GE Healthcare) by calculating the standardized uptake value (SUV) in the regions of interest placed over the suspected lesions using all available clinical information and correlative conventional imaging for anatomic guidance. The SUV was calculated for the quantitative analysis of tumor ¹⁸F-FDG uptake as follows: SUV = C (kBq/mL)/ID (kBq)/body weight (kg), where C is the tissue activity concentration measured by PET and ID is the injected dose.

¹⁸F-FDG uptake was also quantitatively assessed by SUVs calculated in volumes of interest (VOIs) that were placed over regions of abnormal ¹⁸F-FDG uptake. The boundaries of each VOI were checked by comparison with fused CT to exclude adjacent ¹⁸F-FDG avid structures. The maximum SUV (SUV_max) within the VOI was recorded for the primary tumor. Metabolic tumor volume (MTV) was defined as the total tumor volume segmented via the threshold SUV. The threshold of the mediastinal blood pool activity was used to define the lesions. For the threshold SUV established using mediastinal blood pool activity, a VOI of more than 5 × 5 × 5 voxels was drawn manually at the aortic arch. The average SUV at the aortic arch plus two standard deviations of the VOI was adopted as the threshold SUV for the tumor using the mediastinal blood pool. Total lesion glycolysis (TLG) was determined as a product of the average SUV (SUV_mean) segmented via the threshold SUV multiplied by the number of voxels in the MTV (i.e., SUV_mean × MTV). For each patient, we defined the MTV and TLG as the sum of the MTVs and TLGs of all lesions, respectively.

In this study, we investigated the KRAS mutation status in a cohort of 50 consecutive ICC patients who underwent radical hepatectomy and identified KRAS mutations in 32.0% of cases. The following features of our study are significant: (1) correlation between KRAS mutation and glucose uptake is recapitulated in ICC tumors, and (2) metabolic tumor volume on ¹⁸F-FDG-PET may provide useful information as a surrogate for prognosis, reflecting the impact of KRAS mutation on survival.

Malignant tumors can enhance tumor cell survival by genetic changes or modify glucose metabolism by cellular responses [25]. A number of genetic mutations in ICC have been identified [10–14]. In particular, KRAS mutation has been reported as a representative factor indicative of poor prognosis in ICC [12–14], and our patients with mutated KRAS showed significantly worse survival compared to those with wild-type KRAS. Regarding glucose metabolism, Warburg discovered that, even in the presence of oxygen, cancer cells undergo aerobic glycolysis rather than the normal oxidative phosphorylation. Aerobic glycolysis produces just two molecules of adenosine triphosphate (ATP) per molecule of glucose, while 36 ATP molecules are produced by oxidative phosphorylation. Cancer cells have an accelerated metabolism and increased requirements for ATP production. The reason why cancer cells, which need high ATP levels, take this inefficient pathway is not clear. To maintain high ATP levels for energy utilization, cancer cells may increase glucose transport through overexpression of GLUT-1. In this study, 52.0% of patients with ICC showed high expression of GLUT-1, which was associated with KRAS mutation. Whether the upregulation of GLUT-1 is attributable to KRAS mutation or whether GLUT-1 expression contributes to KRAS mutation remains to be established in ICC. High expression of GLUT-1 is associated with multiple tumors and tumor stage, and has been reported as a prognostic factor [26–28]. Patients with high GLUT-1-expressing tumors have a significantly poorer survival compared to patients with low GLUT-1 expression.

It has been reported that ¹⁸F-FDG accumulation reflects the KRAS mutational status of cancers [29–31]. We assessed three parameters measured by ¹⁸F-FDG-PET (SUV_max, MTV, and TLG). In practice, SUV_max is the most commonly assessed parameter of ¹⁸F-FDG-PET, and previous reports have suggested that this parameter is associated with survival in patients with various cancers [32, 33]. Recently, several reports suggested that the volumetric parameters of tumors measured by ¹⁸F-FDG-PET, such as the MTV and TLG, are more accurate prognostic factors than SUV_max in patients with various malignancies [34, 35]. In this study, we have shown that KRAS mutations were significantly associated with high ¹⁸F-FDG uptake as calculated by the MTV and TLG, while SUV_max was comparable between the mutated KRAS group and wild-type group. Volumetric parameters measured by ¹⁸F-FDG-PET have advantages in terms of predicting KRAS mutation status. First, ¹⁸F-FDG-PET is non-invasive and harmless compared to performing a liver tumor biopsy. Second, volumetric parameters reflect the metabolic activity of the entire tumor mass in a three-dimensional manner. There was no association between SUV_max and KRAS mutation in this study, but this could be due to the intratumoral heterogeneity of the KRAS mutation status [13, 36]. SUV_max exhibits only the highest intensity of ¹⁸F-FDG uptake in the tumor and cannot reflect the metabolic activity of the entire tumor. Of the three parameters, the MTV and TLG were associated with KRAS mutation, and ROC analysis showed that the MTV was the best predictor of KRAS mutation.

In this study, the median SUV_max of the tumor lesions was 5.8 (range, 2.9–14.7). No patient with ICC had too little ¹⁸F-FDG uptake to detect the tumor lesion by FDG-PET despite of detectable tumor by CT or magnetic resonance imaging (MRI). When it was difficult to determine boundaries for obscure tumors, the tumor margin was identified using preoperative imaging, such as CT and/or MRI. Morphological size measured by CT or MRI was not significantly different between tumors with mutated KRAS and those with wild-type KRAS. In addition, it is well known that ICC tumors are frequently accompanied by central necrosis as they increase in size. Metabolic volume measured by ¹⁸F-FDG-PET more accurately reflects tumor viability than does radiographic volume, particularly as it takes into account tumor activity.

The current study has a limitation. It was a retrospective design conducted in a single institutional cohort of patients and involved a small study population of 50 consecutive ICC patients, including only 16 patients with KRAS mutation. This weakened the statistical power of our analysis. In addition, patient-selection bias might have influenced the statistical results. However, this study focused exclusively on ICC, rather than on biliary tract cancer, contributing to a better understating of KRAS-related molecular biology. Therefore, this should be considered a preliminary report, and further prospective studies with larger patient cohorts are required to validate the combination of ¹⁸F-FDG-PET parameters in association with somatic mutations and prognosis for patients with ICC.

In this study, we demonstrated that KRAS mutation is associated with GLUT-1 expression and volumetric parameters of ¹⁸F-FDG-PET in ICC tumors. KRAS mutation affects the prognosis of ICC patients undergoing surgical resection and is associated with tumor glucose uptake. Moreover, our results suggest that ¹⁸F-FDG-PET may serve as a potential biomarker for KRAS mutation status and survival in ICC.