Patient Characteristics
A total of 48 PBC patients and 48 PSC patients were selected and matched to separate groups of 48 unaffected controls based on sex, reported race and age at sample collection. Following data generation, one of the PSC patients was found to be an outlier, having liver-specific DNA levels greater than 2-fold higher at each locus than all other patients, and was removed from the study, leaving 47 PSC patients. The characteristics of these patient-control groups are presented in Table 2 (PBC) and Table 3 (PSC). The patient groups were further separated into two groups of 24 patients with early- or late-stage disease based on biochemical and clinical data. These groups were well-matched for most parameters, but PBC patients with late-stage disease were younger at diagnosis than those with early disease, median 42.9 years vs. 52.1 years, respectively, p=0.0185 (Table 2). In PSC this trend was opposite, with advanced disease patients being diagnosed later than patients in the early disease group, median 46.1 years vs. 36.2 years, respectively. However, this difference was not statistically significant, p=0.0693 (Table 3).
Table 2. Characteristics of PBC patients and Controls
|
|
Controls
(n=48)
|
PBC (all)
(n=48)
|
p-val†
|
PBC (early)
(n=24)
|
PBC (late)
(n=24)
|
p-val‡
|
Sex, %Male
|
12.5
|
12.5
|
>0.9999
|
8.3
|
16.7
|
0.6662
|
Race, %Caucasian
|
98
|
91.7
|
0.3616
|
95.8
|
87.5
|
0.6085
|
Age at sample collection (yrs), median (IQR)
|
56.3 (48.2-61.0)
|
56.3 (41.3-56.5)
|
0.9927
|
57.8 (53.3-62.2)
|
51.7 (46.5-61.0)
|
0.1071
|
ALP (xULN), median (IQR)
|
0.60 (0.48-0.73)
|
1.17 (0.83-4.02)
|
<0.0001
|
0.83 (0.71-0.94)
|
3.98 (3.17-5.33)
|
<0.0001
|
AMA,
(% positive)
|
0
|
81.3
|
<0.0001
|
79.2
|
83.3
|
>0.9999
|
Age at Dx (yrs), median (IQR)
|
na
|
49.4 (41.3-56.5)
|
-
|
52.1 (46.3-57.4)
|
42.9 (39.9-51.9)
|
0.0185
|
Disease duration (yrs), median (IQR)
|
na
|
5.0 (2.0-9.0)
|
-
|
4.5 (2.0-9.0)
|
5.5 (2.0-10.8)
|
0.6116
|
Clin. FU (yrs), median (IQR)
|
na
|
6.0 (4.0-10.0)
|
-
|
8.5 (5.3-10.0)
|
5.0 (2.0-8.8)
|
0.0133
|
UDCA treatment, (%)
|
na
|
97.9
|
-
|
100
|
95.8
|
>0.9999
|
†p-value for control vs. PBC (all) comparison, ‡p-value for PBC (early) vs. PBC (late) comparison; ALP (xULN): alkaline phosphatase expressed as times the upper limit of normal, AMA: anti-mitochondrial antibodies, Clinical FU: clinical follow-up after sample collection, UDCA: ursodeoxycholic acid
|
Table 3. Characteristics of PSC patients and Controls
|
|
Controls
(n=48)
|
PSC (all)
(n=47)
|
p-val†
|
PSC (early)
(n=24)
|
PSC (late)
(n=23)
|
p-val‡
|
Sex, %Male
|
58.3
|
59.6
|
>0.9999
|
62.5
|
56.5
|
0.4158
|
Race, %Caucasian
|
100
|
98.0
|
0.4947
|
95.8
|
100
|
0.6085
|
Age at sample collection (yrs), median (IQR)
|
52.3 (44.1-62.1)
|
54.3 (32.8-62.1)
|
0.4847
|
47.0 (29.8-58.7)
|
57.4 (42.5-63.5)
|
0.1599
|
ALP (xULN), median (IQR)
|
0.59 (0.47-0.72)
|
1.03 (0.86-3.65)
|
<0.0001
|
0.86 (0.74-0.95)
|
3.65 (2.96-5.36)
|
<0.0001
|
Total bilirubin,
median (IQR)
|
na
|
0.8 (0.5-1.8)
|
-
|
0.6 (0.5-0.9)
|
1.6 (0.8-3.4)
|
<0.0001
|
Age at Dx (yrs), median (IQR)
|
na
|
42.4 (28.2-53.9)
|
-
|
36.2 (22.0-48.5)
|
46.1 (34.9-57.5)
|
0.0693
|
IBD Type
|
na
|
|
-
|
|
|
0.3947
|
UC, n
|
|
36
|
|
18
|
18
|
|
CD, n
|
|
2
|
|
2
|
0
|
|
Ind. IBD, n
|
|
3
|
|
2
|
1
|
|
None, n
|
|
6
|
|
2
|
4
|
|
Disease duration (yrs), median (IQR)
|
na
|
6.0 (3.0-12.0)
|
-
|
5.0 (2.0-12.5)
|
6.0 (3.0-12.0)
|
0.8035
|
Clin. FU (yrs), median (IQR)
|
na
|
5.0 (3.0-6.0)
|
-
|
5.0 (4.0-6.8)
|
4.0 (1.0-6.0)
|
0.0699
|
UDCA treatment, (%)
|
na
|
70.2
|
-
|
69.6
|
77.3
|
0.7381
|
†p-value for control vs. PSC (all) comparison, ‡p-value for PSC (early) vs. PSC (late) comparison; ALP (xULN): alkaline phosphatase expressed as times the upper limit of normal, IBD: inflammatory bowel disease, UC: ulcerative colitis, CD: Crohn’s disease, Ind. IBD: Indeterminate IBD, Clin. FU: clinical follow-up after sample collection, UDCA: ursodeoxycholic acid
|
Assay Performance
We found that the multiplex amplicon-based method provides for very high read counts at each CpG site, with median counts in the range of 10,000 for CpGs in the VTN amplicon and over 30,000 for CpGs in the ITIH4 and IGF2R amplicons (Table 1). The ratios of unmethylated to methylated CpGs were relatively consistent in the CpG sites at IGF2R and VTN across the study population, with median values ranging from 0.030-0.041 and 0.045-0.064, respectively (Table 1). However, the unmethylated ratios of CpGs in ITIH4 were more variable, with one of the CpGs, ITIH4-1, being significantly higher than other evaluated CpGs with a median unmethylation ratio value of 0.280 (Table 1). This suggests either an assay-based artifact or that ITIH4-1 unmethylation may not truly be liver-specific, and thus, it was removed from the analysis. Liver-specific DNA concentrations in our controls seemed to be higher than those in the original report,(12) possibly due to minor technical differences in the assay used. Consistent with the previous report(12), we did not detect an influence of age on liver-specific DNA concentration in controls (Figure 1A). Likewise, age did not influence liver-specific DNA concentration in PBC (Figure 1B) or PSC (Figure 1C) patients. Finally, we found that sex did not influence liver-specific DNA levels as measured by all 3 genes: IGF2R (Figure 2A), ITIH4 (Figure 2B) and VTN (Figure 2C).
Liver-specific circulating cfDNA is increased in PBC and PSC patients compared to controls and in late-stage compared to early-stage disease
The liver-specific circulating cfDNA (Geq/ml) values were used to make comparisons between patient and control groups and between patients with early- and late-stage disease. Results of these analyses are shown in Figure 3. PBC patients demonstrated significantly increased liver-specific DNA compared to controls as measured by all 3 genes, IGF2R: (median (IQR)) 281.93 (126.24-597.61) Geq/ml vs. 112.85 (66.71-184.19) Geq/ml, p=0.0001 (Figure 3A), ITIH4: 441.79 (256.40-837.79) Geq/ml vs. 256.83 (122.99-399.69) Geq/ml, p=0.0004 (Figure 3B), and VTN: 334.73 (149.76-879.57) Geq/ml vs. 133.13 (72.03-202.66), p<0.0001 (Figure 3C). These levels were also significantly increased in PBC patients with late-stage compared to early-stage disease when measured by IGF2R: (median (IQR)) 453.79 (221.47-862.67) Geq/ml vs. 180.20 (95.97-355.83) Geq/ml, p=0.0068 (Figure 3A) and VTN: 822.01 (248.81-1359.55) Geq/ml vs. 198.29 (114.34-595.37), p=0.0024 Geq/ml (Figure 3C). However, the increase when measured using ITIH4 was not statistically significant (median (IQR)) 539.26 (281.77-1273.28) Geq/ml vs. 364.84 (225.98-581.50), p=0.0701 (Figure 3B).
PSC patients also demonstrated significantly increased liver specific DNA compared to controls as measured by all 3 genes, IGF2R: (median (IQR)) 317.46 (96.93-479.11) Geq/ml vs. 122.10 (35.65-165.06) Geq/ml, p<0.0001) (Figure 3D), ITIH4 475.76 (124.81-929.76) Geq/ml vs. 247.23 (117.87-470.36) Geq/ml, p=0.0066) (Figure 3E), and VTN: 415.12 (170.68-1000.30) Geq/ml vs. 184.02 (53.83-285.65) Geq/ml, p<0.0001) (Figure 3F). These levels were also significantly increased in PSC patients with late-stage compared to early stage disease when measured by ITIH4 (median (IQR)): 759.85 (189.59-1377.80) Geq/ml vs. 318.34 (121.44-566.00) Geq/ml, p=0.0177 (Figure 3E) and VTN: 867.24 (194.71-1227.01) Geq/ml vs. 307.94 (159.98-427.80) Geq/ml, p=0.0138 (Figure 3F). However, the increase when measured using IGF2R was not quite statistically significant (median (IQR)) 443.17 (86.90-862.92) Geq/ml vs. 257.40 (105.79-360.62), p=0.0563 (Figure 3D).
Liver-specific circulating cfDNA levels are correlated with alkaline phosphatase levels in PBC and PSC patients but not in controls
Liver function tests, particularly ALP, are often used to evaluate liver damage and disease severity in cholestatic liver diseases such as PBC and PSC.(20, 21) Thus, we evaluated the potential correlation between liver-specific circulating cfDNA and ALP (expressed as times the ULN) using the Pearson correlation coefficient. The results of these analyses are presented in Figure 4 and show significant correlation between ALP and cfDNA levels as measured by all 3 genes in PBC (Figure 4A) and PSC (Figure 4B) but not in controls (Figure 4C). We also had data available for Total bilirubin, another commonly used liver function test, in the PSC patients and found that those values did not correlate with liver-specific DNA levels as measured by any of the 3 genes (Figure 4D).