The detailed process of IDS
Preoperative MRI showed that the tumor was located in the posterior lower segment of the right lobe of the liver. The size of the tumor was 72mm*58mm*55mm. T2W1 showed slightly higher, equal, and uneven equal-high signals. Scanning after enhancement, the hepatic artery phase demonstrated obvious uneven enhancement. In the portal phase and delayed phase, the relative signal attenuation was in line with the primary liver cancer (Fig. 2A). The preoperative evaluation was in accordance with the inclusion criteria. During the operation, the tumor was completely resected (R0 resection), and the whole specimen was taken to produce macro-slide. After hematoxylin-eosin staining, the macro-slide was observed at different positions with various magnifications (Fig. 2B). Then, the pathological macro-slide was matched with tumor specimen and imaging data to obtain WSI including MVI positions (Fig. 2C). This total process was named as IDS.
The baseline characteristics and long-term outcomes of eligible patients
A total of 110 primary liver cancer patients were collected in this study. 19 patients who were pathologically diagnosed as intrahepatic cholangiocarcinoma were excluded, and 91 HCC patients were finally included. The baseline clinicopathological characteristics were shown in Table 1. Almost all patients had hepatitis B or C virus infection background. The percentage of hepatitis B virus infection was 96.7%, and only one patient did not had hepatitis. The liver function of patients was all graded as Child-Pugh class A. The median tumor size was 3.80 cm. In 4 HCC patients associated with portal vein tumor thrombus (PVTT), PVTT existed in the branches of main portal vein and could be resected radically. The shortest and longest follow-up times were 13 and 28 months, respectively. 24 (26.37%) patients developed disease recurrence and 5 patients (5.49%) died during follow-up.
MVI detection rates in 3-Point, 7-Point baseline sampling protocols and IDS
As shown in Fig. 3A, the detection rates of MVI were 21.98%, 32.97% and 63.74%, respectively, in 3-Point, 7-Point and IDS (P< 0.001). Patients with MVI positive status in 3-Point and 7-Point were all included in MVI positive status in IDS. The populations of 3-Point and 7-Point were not exactly the same (Fig. 3B). Among patients with MVI negative status in 3-Point and 7-Point, the two populations were partly different, but they all included MVI negative status in IDS (Fig. 3C). Therefore, in this study, the specificity and sensitivity of IDS on MVI detection were both 100%, while the specificity of 3-Point and 7-Point on MVI detection was both 100%, and the sensitivity of 3-Point and 7-Point on MVI detection was only 34% and 52%, respectively (Table 2). The above results showed that IDS had superior sensitivity and specificity for the detection of MVI than 3-Point and 7-Point.
Tumor recurrence rates in three various pathological examination methods
To compare the impact of MVI, which was detected by 3-Point, 7-Point and IDS, respectively, on tumor recurrence, survival analyses were performed (Fig. 4A–C). Under the three methods, patients with MVI positive status were more likely to relapse (P< 0.001, P< 0.001, P= 0.001, respectively). We found that 12 (16.90%) HCC patients with MVI negative status in 3-Point had tumor recurrence, and 2 patients died due to disease progression. In 7-Point sampling method, 10 (16.39%) patients with MVI negative status had recurrence (relapse time range: 2.1–14.4 months), including 2 recurrence-related deaths. In IDS, 2 (6.06%) patients with MVI negative status recurred (relapse time: 13.13 and 14.40 months), and no deaths occurred. Next, we did subgroup analysis combining IDS with 3-Point and 7-Point. Results showed that patients with MVI positive status in both 3-Point and IDS, and in both 7-Point and IDS were most prone to recur. Patients with MVI positive status in IDS were more likely to relapse than patients with actual MVI negative status (P= 0.021, P= 0.016). (Fig. 4D–E). It is to say, 3-Point and 7-Point sampling protocols have a potential possibility to miss MVI, and patients with MVI false negative status in 3-Point and 7-Point are more likely to recur than patients with actual MVI negative status in all three MVI pathological testing methods.
Identification of potential biomarkers to distinguish MVI false negative patients in conventional pathological sampling protocols
In order to find out the clinicopathological characteristics of patients with missed MVI in 3-Point and 7-Point, IDS was matched with 3-Point and 7-Point. As shown in Table 3, There were significant differences in 3-Point in AFP, PIVKA-II, ALP, and tumor number. The medians of AFP between 3-Point negative IDS negative and 3-Point negative IDS positive groups were 6.10 (3.10, 20.30) ug/L and 81.30 (10.12, 560.65) ug/L. The medians of PIVKAII between 3-Point negative IDS negative and 3-Point negative IDS positive groups were 107.00 (33.00, 412.00) mAU/mL and 449.00 (90.50, 3168.75) mAU/mL. The medians of ALP between 3-Point negative IDS negative and 3-Point negative IDS positive groups were 79.00 (63.00, 92.00) U/L and 64.50 (54.25, 79.25) U/L. As shown in Table 4, only AFP and PIVKA-II had significant differences in 7-Point. The medians of AFP between 7-Point negative IDS negative and 7-Point negative IDS positive groups were 6.10 (3.10, 20.30) ug/L and 160.55 (20.92, 1210.00) ug/L. The medians of PIVKA-II between 7-Point negative IDS negative and 7-Point negative IDS positive groups were 107.00 (33.00, 412.00) mAU/mL and 460.50 (239.25, 3084.25) mAU/mL, respectively. It revealed that AFP and PIVKA-II could be potential biomarkers to distinguish MVI false negative patients in 3-Point and 7-Point.
Comparison of sensitivity and specificity of AFP and PIVKA-II in identifying MVI false negative patients
In order to study the sensitivity and specificity of AFP and PIVKA-II to identify MVI false negative patients, 71 patients with MVI positive and negative status in IDS but both negative in 3-Point were compared, and a total of 61 patients with MVI positive and negative status in IDS but both negative in 7-Point were compared. After calculating the best cutoff by maximizing the Youden index (Table 5), in 3-Point, when AFP was divided by a cutoff of 22.5 ng/mL, the AUC was 0.715 (0.592–0.837), the sensitivity was 0.68 (0.51–0.82), and the specificity was 0.79 (0.61–0.91). When using 267 mAU/mL as the cutoff of PIVKA-II, the AUC was 0.665 (0.538–0.793), the sensitivity was 0.66 (0.49–0.80), and the specificity was 0.67 (0.48–0.82) (Fig. 5A–B). In 7-Point, when AFP was divided by a cutoff of 23.9 ng/mL, the AUC was 0.748 (0.617–0.879), the sensitivity was 0.75 (0.55–0.89), and the specificity was 0.79 (0.61–0.91). When using 267 mAU/mL as the cutoff of PIVKA-II, the AUC was 0.696 (0.558–0.833), the sensitivity was 0.75 (0.55–0.89), and the specificity was 0.67 (0.47–0.81) (Fig. 5C–D). Therefore, AFP is superior to PIVKA-II as a biomarker to distinguish MVI false negative patients.
Potential clinical utility of upper limit of AFP normal value to identify MVI false negative patients in conventional pathological sampling protocols
Because the cutoff values of AFP in 3-Point and 7-Point (22.5 and 23.9 ng/mL) were similar to the upper limit of normal value of AFP (20 ng/mL), the comparison and analysis were carried out using 22.5 ng/mL and 20 ng/mL, 23.9 ng/mL and 20 ng/mL respectively. In 3-Point sampling method, the detection rate of patients with MVI false negative status was 68.4% (26/38), and there was a significant difference (Fig. 5E, Table 6). In 7-Point sampling method (Fig. 5F, Table 7), the detection rates of patients with MVI false negative status patients were 75% (21/28) and 70% (21/30) respectively, both of which were significantly different. That is to say, in 3-Point and 7-Point, 68.4% and 70% of patients with MVI negative status and with AFP greater than 20 ng/mL are likely to be false negative.