2.1 Demographic characteristics
Of the 440 patients, 214 were diagnosed with BA, and 226 non-BA jaundice patients were recognized as the control group. All included patients were followed up to confirmation of diagnoses, and the diagnoses of 440 patients were listed in Supplementary Table 1. The baseline demographic, clinical, and laboratory profiles of the included patients were shown in Table 1.
2.2 Optimal threshold value and age-related changes in serum MMP-7 levels
In neonate cholestasis patients (n = 65; BA = 34, non-BA = 31), using a cut-off value of > 26.73 ng/ml, serum MMP-7 had a sensitivity = 91.2%, specificity = 100%, and AUC of 0.954 [95% CI: 0.897-1.000]. (Supplementary Figure 1) In order to reduce missed opportunities for early BA diagnosis in cholestasis patients, we examined the diagnostic accuracy of using > 26.73 ng/mL in all patients without age limit (n = 440; BA = 214, non-BA = 226). Using a cut-off value of > 26.73 ng/mL in all cholestasis patients, serum MMP-7 had a sensitivity = 96.7%, specificity = 95.6%, and AUC of 0.983 [95%CI: 0.970-0.996]. The positive predictive value (PPV), negative predictive value (NPV), and diagnostic efficiency of serum MMP-7 level >26.73 ng/mL for predicting BA were 95.39%, 96.86%, and 96.14%, respectively. (Supplementary figure 2)
To investigate whether MMP-7 was age-related, we divided the included patients into three age groups (Group A: 4-28 days, n = 65, Group B: 29-60 days, n = 234, Group C: ≥ 61 days, n = 141). Analysis based on age groups showed that BA patients in group C had significantly higher serum MMP-7 levels than those in group A and group B. No significant differences were identified between group A and group B. In non-BA patients, no age-related differences were noted (Supplementary figure 3).
2.3 Reasons for low serum MMP-7 level in BA patients
Although serum MMP-7 has high diagnostic accuracy, 7 (3.27%) BA patients in our cohort had serum MMP-7 lower than 26.73ng/ml. To assess the reason for the low MMP-7 expression level in these BA patients, we performed polymorphism sequencing on exons and promoter regions of MMP-7. The heterozygote mutation percentage of c.410G >A (rs17884789 C > T) in exon 3 was significantly increased in LSM patients compared with HSM patients (30.00% vs 3.03%, P=0.032). (Supplementary Table 2)
As a small molecular weight protein, MMP-7 showed relatively poor stability in vitro. Since all serum samples were centralized to one center for testing, a standard transportation process should be developed.
How different transportation environments impacted serum MMP-7 levels were simulated to reduce the influence of external factors during transportation. MMP-7 degradation rates under different conditions (room temperature, 4°C, and dry ice) are presented in Supplementary Figure 4. The level of MMP-7 was stable in both whole blood and serum on dry ice. In the 4°C refrigerators, serum MMP-7 protein levels were reduced to less than 90% after 12 h (whole blood) and 48 h (serum). At room temperature (RT), whole blood and serum MMP-7 degradation exceeded 50% after 4 hours (Supplementary Figure 4A). Each freezing-thawing procedure resulted in an approximately 15% reduction in MMP-7 serum levels (Supplementary Figure 4B).
2.4 Correlation of serum MMP-7 levels with multiple clinical characteristics at diagnosis
A total of 191 of the 214 BA patients underwent surgery, and liver biopsies were obtained. Ten patients with histological cirrhosis and decompensation were referred to other departments for liver transplantation, and 13 patients did not have consent for KPE. Hence, a total of 191 cases were included for the following correlation analysis. Serum MMP-7 levels at diagnosis were found to positively correlate with total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT) at diagnosis and fibrosis classification and inflammation grade at KPE in BA patients (Supplementary Table 3). The multivariable linear regression model examining serum MMP-7 levels is summarized in Supplementary Table 4. The γ-GT level at diagnosis and inflammation grades and fibrosis classifications at KPE were significant factors associated with serum MMP-7 levels at diagnosis. Moreover, MMP-7 showed a significant mediation effect on the association between inflammation and liver fibrosis in BA infants; however, no mediation effect was identified in non-BA infants (Table 2). The result indicated that MMP-7 could be a critical factor associated with inflammation-induced fibrogenesis in BA.
2.5 Dynamic changes in serum MMP-7 in BA patients post-KPE
Eighty-three BA patients who had a follow-up of less than two-year or were lost to follow-up after discharge were therefore excluded from the dynamic trend analysis. No significant differences were identified between the demographics of the included and excluded patients and SNL percentages of each centre (Supplementary table 5, 6). Hence, 108 BA patients were included for the following analysis. 62 (57.41%) and 51 (47.22%) post-KPE BA patients had event-free survival with their native liver (SNL) during the first and second-year post-KPE, respectively. Four patterns of changes in serum MMP-7 levels post-KPE were observed. Detailed dynamic post-KPE serum MMP-7 changes in four representative patients are shown in Figure 2. MMP-7 pattern one (n = 11, 10.19%) involved a rapid decline and then gradually increased or remained relatively stable at a low level (below the postoperative level), finally presenting a downward trend (Figure 2A). MMP-7 pattern two (n =29, 26.85%) showed elevated or fluctuating MMP-7 levels around a relatively high level and then a stable downward trend (Figure 2B). MMP-7 pattern three (n = 51, 47.22%) involved a drastic fluctuation in the first 1-2 months post-KPE and subsequently elevated MMP-7 levels, even though the jaundice-free status ever achieved post-KPE (Figure 2C). MMP-7 pattern four (n =17, 15.74%) involved a continuously rising serum MMP-7 level post-KPE and usually had persistent jaundice (Figure 2D). The percentage of SNL patients at the two-years visit in the dynamic pattern one to four is 100%, 79.31%, 31.37%, and 5.88%, respectively. The dynamic trends of serum MMP-7 levels in BA patients post-KPE with different prognoses (SNL vs. non-SNL) are summarized in Figure 2E. Significant differences between the two groups began to appear from four weeks post-KPE.
To determine the reason for the rapid decline in serum MMP-7 levels (pattern one) in some BA patients post-KPE, we further analysed MMP-7 levels in stool samples of 40 post-KPE BA patients. In response to the two changing directions (increase/decrease) in serum MMP-7 levels, two stool MMP-7 level dynamic patterns were observed. The detailed post-KPE serum and stool MMP-7 levels of two representative patients are shown in Figure 2 F, G. Patient 1 had a rapidly increased stool MMP-7 level with a decreasing/fluctuating serum MMP-7 level (Stool pattern 1, n = 17, Figure 2F). Patient 2 had a stable low stool MMP-7 level; however, her serum MMP-7 level increased continuously after KPE (stool pattern 2, n = 3, Figure 2G). The stool MMP-7 levels were almost undetectable in BA patients at diagnosis. SNL patients had significantly higher stool MMP-7 levels post-KPE than LTx patients. (Supplementary Table 7)
2.6 Correlation of serum MMP-7 levels with multiple clinical characteristics at 3 months post- KPE
Supplementary Table 8 shows the positive correlations between postoperative clinical indicators and serum MMP-7 levels at three months post-KPE. The multivariable linear regression model showed that total bilirubin, Fibro Touch results and APRI were significantly associated with serum MMP-7 levels at three months post-KPE (Supplementary Table 9).
2.7 The power of postoperative clinical indicators to predict SNL at two years post-KPE
The average MMP-7 levels at diagnosis were not different between BA patients who survived with native liver (SNL) or not at two years post-KPE (68.95, IQR: 55.73 – 106.26 ng/ml; vs. 74.43, IQR: 58.57 – 96.13 ng/ml, p = 0.808). Age was the only preoperative indicator identified to have predictive power for SNL at two years post-KPE (SNL: 43.96 ± 2.93 days vs. non-SNL: 57.49 ± 3.79 days, p = 0.007). Serum MMP-7 was the only indicator with significant differences at six weeks post-KPE, while all listed postoperative indicators at three months post-KPE showed significant differences (Supplementary Table 10). The ROC analyses showed varying degrees of power for serum MMP-7 levels, total bilirubin, direct bilirubin, γ-GT, Fibro Touch levels, and APRI post-KPE in predicting SNL at two years post-KPE (Figure 3) and MMP-7 at three months post-KPE having the highest AUC (0.8612, 95% CI: 0.790-0.932). Delong’s test showed that serum MMP-7 level at six weeks and three months post-KPE has the highest prognostic value among all current minimally invasive indicators (Supplementary Table 11). However, the prediction ability of serum MMP-7 did not increase when combined with other indicators at six weeks post-KPE.
We also employed Kaplan-Meier survival analysis and Cox regression to ascertain which factors had strong correlations with SNL at two years post-KPE in BA patients. Figure 4 illustrates survival curves with log-rank tests for MMP-7 > 75.22 ng/ml, TBIL > 35.6 µmol/L, DBIL > 18.9 µmol/L, GGT > 306.1 U/L, APRI > 2.5448, and Fibro Touch > 19.35 at 3 months post-KPE. The Cox regression results showed that serum MMP-7 level, total bilirubin, and APRI at 3 months after KPE were significant indicators of SNL at two years post-KPE and that serum MMP-7 level had the highest hazard ratio (5.34, 95%CI [2.50 – 11.39]) (Supplementary Table 12).