Protein Induced by Vitamin K Absence or Antagonist-II: Significantly Elevated in Obstructive Jaundice and Sepsis Patients Without Hepatocellular Carcinoma.

1
Introduction
Liver cancer represents a significant global health challenge, with projections indicating that it will affect more than 1 million individuals annually by 2025. Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, accounts for approximately 90% of cases and is the fourth leading cause of cancer‐related death worldwide. Given its high mortality rate, early detection through screening programs is essential, as timely diagnosis enables effective treatment and improves long‐term disease‐free survival [1, 2].
Current guidelines recommend performing abdominal ultrasound every 6 months for the surveillance of patients with cirrhosis. However, it has been established that ultrasound is insufficiently reliable for detecting hepatocellular carcinoma at an early stage, with a sensitivity of only 63% in this setting [3, 4]. Thus, additional biomarkers for early HCC detection are needed to complement ultrasound. Alpha‐fetoprotein (AFP) is the most widely used serum marker for HCC surveillance globally. Previous studies have reported that serum AFP demonstrates a sensitivity of 39%–65% and a specificity of 76%–94% for detecting HCC, which remains suboptimal for clinical application. Protein induced by vitamin K absence or antagonist‐II (PIVKA‐II), also called des‐c‐carboxy prothrombin (DCP) initially identified in 1984 as a specific biomarker for HCC [5], has emerged as a promising diagnostic and prognostic tool for HCC surveillance, including in early‐stage and AFP‐negative disease [6, 7]. Moreover, elevated PIVKA‐II serum levels and increased tissue expression have been associated with microvascular invasion [8], a major risk factor for tumor recurrence and mortality in HCC, though a consensus definition remains lacking [9].
Prior evidence has established the role of PIVKA‐II in screening and diagnosing HCC. However, it should be noted that PIVKA‐II can be elevated even in the absence of HCC, particularly among populations targeted for surveillance and pending diagnosis. PIVKA‐II levels are elevated in the presence of vitamin K deficiency [5, 10]. Additionally, antibiotics containing an N‐methylthiotetrazole substrate, obstructive jaundice (associated with pancreatic head cancer, biliary tract cancer, or benign stricture of the bile duct), alcohol abuse, and warfarin ingestion can lead to false positive PIVKA‐II results [10, 11, 12]. Therefore, we conducted a study involving patients with obstructive jaundice or sepsis to investigate the distribution and positive rates of PIVKA‐II and AFP and their relationships with laboratory tests.

2
Materials and Methods
2.1
Patient
This cross‐sectional study included patients with obstructive jaundice and sepsis, who were recruited from the departments of infectious diseases, respiratory and critical care medicine, interventional radiology, and hepatobiliary surgery at Wuhan Union Hospital. The study was approved by the Ethics Committee of Wuhan Union Hospital (the approved number 0887).
The obstructive jaundice cohort (n = 58) included patients with the following underlying conditions: pancreatic head cancer (n = 8), cholangiolithiasis (n = 4), cholangiocarcinoma (n = 40), cholestatic hepatitis (n = 4), and gallbladder adenocarcinoma (n = 2). Among these, 24 patients received multivitamin supplementation during hospitalization. Patients with sepsis (n = 55) who received antibiotics containing N‐methylthiotetrazolium substrates included pneumonia (n = 18), liver abscess (n = 40), and abdominal infection (n = 2), of which five cases had pneumonia and liver abscess co‐infection. Of these, 25 patients received multivitamin supplementation during hospitalization. Patients with liver cirrhosis, blood diseases, primary hepatocellular carcinoma, mixed primary liver cancer, metastatic hepatocellular carcinoma and severe liver dysfunction were excluded. A total of 241 patients with primary hepatocellular carcinoma and 125 healthy individuals were selected as the control group (Figure 1).

2.2
Measurements
Individuals included underwent blood sampling for liver biochemical tests, including ALT, AST, ALP, GGT, albumin, bile acids, total bilirubin, and conjugated bilirubin, as well as inflammatory markers (C‐reactive protein, procalcitonin), coagulation function parameters (PT, INR, APTT), blood cell counts (WBC, neutrophils), and tumor markers (PIVKA‐II, AFP). All measurements were conducted using standardized laboratory techniques at Wuhan Union Hospital.
Serum AFP and PIVKA‐II concentrations were measured by chemiluminescence microparticle immunoassay using the Abbott Architect i system. In this study, the established normal cut‐off values for these makers were as follows: AFP < 7.78 ng/mL and PIVKA‐II < 40.0 mAU/mL.

2.3
Statistical Analysis
Laboratory test concentrations were characterized using the median and interquartile range (IQR). Variables without a normal distribution were analyzed using the Mann–Whitney U test. Categorical parameters were evaluated using either the Chi‐Square test or Fisher's Exact Test. The statistical analyses were performed using SPSS software (version 22). Pearson correlations were determined among PIVKA‐II, AFP, and other variables. For the purpose of analysis, all laboratory values below or above the limit of detection for a particular assay were recorded as the lowest or highest value for that assay. All p values were two‐sided.

3
Results
One hundred and twenty‐five healthy individuals and 354 patients were enrolled in this study, including 58 with obstructive jaundice, 55 with sepsis, and 241 with primary hepatocellular carcinoma. Laboratory values for the study cohort are summarized in Table 1.
To evaluate whether markers of the severity of obstructive jaundice such as bile acids, total bilirubin, conjugated bilirubin, and coagulation function indicators (PT, INR, and APTT) were associated with serum PIVKA‐II, Pearson correlations were conducted between PIVKA‐II, AFP, and other measured parameters. Serum PIVKA‐II in patients with obstructive jaundice was significantly correlated with PT, INR, and APTT (p < 0.001, p < 0.001, and p = 0.002, respectively), as well as the severity of obstructive jaundice, as measured by serum total bilirubin and conjugated bilirubin (p = 0.018 and p = 0.049) (Table S1, Figure 2). In patients with sepsis, serum PIVKA‐II was positively correlated with PT, INR, and PCT (p = 0.029, p = 0.041, and p < 0.001, respectively). In contrast, no significant correlations were observed between serum AFP and coagulation function indicators. However, positive correlations were observed between AFP and ALT, AST, and bile acids (p < 0.001, p = 0.006, and p = 0.022, respectively) in obstructive jaundice, and between AFP and WBC, neutrophils, and CRP (p < 0.001, p = 0.001, and p = 0.005, respectively) in sepsis (Table S2, Figure 2). Notably, no significant correlations were found between serum PIVKA‐II and AFP in patients with obstructive jaundice and sepsis (p = 0.203 and p = 0.422). Serum PIVKA‐II was positively correlated with AFP, ALT, AST, ALP, and GGT in patients with HCC (r = 0.187, p = 0.004; r = 0.201, p = 0.002; r = 0.237, p < 0.001; r = 0.293, p < 0.001; and r = 0.364, p < 0.001, respectively), but not with PT, INR, and APTT (r = 0.029, p = 0.651; r = 0.019, p = 0.771; and r = 0.090, p = 0.162, respectively) (Table S3, Figure 2).
As shown in Figure 3, there was no significant difference in PIVKA‐II concentration between the HCC and obstructive jaundice groups. However, statistical differences were observed in comparisons among all other groups (p < 0.001). Similarly, the AFP levels showed significant differences among all groups except for sepsis patients and healthy controls (p < 0.001). The seropositivity rate of serum PIVKA‐II was significantly higher than that of AFP across all three groups. Specifically, the seropositivity of PIVKA‐II in patients with obstructive jaundice was 82.76%, which was even higher than that observed in patients with primary hepatocellular carcinoma (75.10%) (Table 2).
The receiver operating characteristic (ROC) curve was used to determine the specificity and sensitivity of PIVKA‐II and AFP in diagnosing HCC under the interference of obstructive jaundice and sepsis (Figure 4). When the cut‐off for PIVKA‐II and AFP was 5.90 ng/mL and 42.17 mAU/mL, respectively, the Youden index (YI) for HCC was the best (Table 3). However, in the presence of sepsis and obstructive jaundice, the specificity, YI, positive likelihood ratio (+LR), and positive predictive value (PPV) of PIVKA‐II were inferior to AFP.
In the obstructive jaundice cohort, there are 50 patients diagnosed with cancer and 40 cases are cholangiocarcinoma. There is no difference in PIVKA‐II levels and positive rates between benign obstructive jaundice and malignancy‐associated jaundice (p = 0.107 and p = 0.131) (Table 4). In patients with obstructive jaundice and sepsis, no significant difference in multivitamin uptake rates was found between the high‐PIVKA‐II and low‐PIVKA‐II groups (p = 0.922 and p = 0.162) (Table 5).

4
Discussion
Normal liver produces prothrombin under vitamin K action, but abnormal prothrombin is produced in patients with vitamin K deficiency or HCC. Protein induced by vitamin K absence or antagonist‐II, also called des‐c‐carboxy prothrombin is an abnormal form of prothrombin, resulting from disruptions in vitamin K metabolism or impaired γ‐glutamyl carboxylase activity in HCC, leading to the absence of γ‐carboxylated amino acid residues. PIVKA‐II is an additional serum marker for AFP‐negative or early‐stage HCC [13, 14].
In the present study, we found that positive rates of PIVKA‐II were significantly higher than those of AFP in patients with obstructive jaundice, sepsis and hepatocellular carcinoma in China, among which patients with obstructive jaundice had the highest positive rate of PIVKA‐II, and there was no difference in PIVKA‐II levels between obstructive jaundice cases and patients with HCC. Furthermore, serum PIVKA‐II was positively associated with AFP in patients with primary HCC, but no such association was observed in patients with obstructive jaundice and sepsis. This discrepancy may be due to the fact that both AFP and PIVKA‐II were associated with hepatocellular carcinoma in primary HCC patients, thereby establishing a correlation. In contrast, in patients with obstructive jaundice and sepsis, PIVKA‐II was primarily influenced by vitamin deficiency, especially in obstructive jaundice, while AFP was not associated with the vitamin and there was no link between them.
A pilot study in Italy showed that PIVKA‐II was positive in 24/26 (92.31%) of the pancreatic cancer patients, and its median serum concentration was also significantly higher than that of benign pancreatic disease [15]. Using two PIVKA‐II antibodies, Kanazumi et al. [16] found elevated PIVKA‐II levels were also observed in 10/27 (37.0%) and in 15/27 (55.6%) of the patients with pancreatic cancer, 7 of 16 (43.8%) and 9 of 16 (56.3%) of those with biliary tract cancer. The high levels of PIVKA‐II in patients with pancreatic cancer and biliary tract cancer were consistent with our findings.
A systematic review of 38 studies with 11,124 cases, revealed that PIVKA‐II alone was moderately accurate in detecting HCC (sensitivity 0.66, 95% CI 0.65–0.68; specificity 0.88, 95% CI 0.87–0.90; positive likelihood ratio [+LR] 7.13, 95% CI 5.73–8.87) [17]. However, in this study, the sensitivity, specificity, and +LR of PIVKA‐II in diagnosing hepatocellular carcinoma were 74.69%, 70.17%, and 2.51, respectively. The specificity and +LR in this study were inferior to measures in other studies. The reason was the increase in PIVKA‐II caused by sepsis and obstructive jaundice.
In this study, prolonged PTs in patients with obstructive jaundice and sepsis were significantly associated with elevated PIVKA‐II levels, but no such correlation was found in patients with primary hepatocellular carcinoma. Increased undercarboxylated prothrombin or decreased vitamin K levels have previously been identified in adults with cholestatic liver disease [18]. Similarly, Strople et al. [19] reported that PIVKA‐II was directly associated with PT and conjugated bilirubin in cholestatic liver disease, which strongly suggested that abnormal PIVKA‐II was the result of vitamin K deficiency. Excluding primary hepatocellular carcinoma, determination of prothrombin time and measurement of PIVKA levels (especially PIVKA‐II, or degamma‐carboxylated prothrombin) can be used to assess the severity of vitamin K deficiency [19, 20]. Impaired coagulation in patients with obstructive jaundice includes damage to liver cells, bacterial ectopia, and a lack of bile in the gut leading to poor absorption of vitamin K. Beyond septic and inflammatory complications, which contribute to hypercoagulability, the underlying pathology involves frequent antibiotic use, which disrupts the gut microbiota and inhibits the production of vitamin K2 by gut bacteria [20, 21, 22, 23]. Poor nutrition due to inadequate intake of hospital food, changed the intestinal microflora as a result of antibiotic treatment are the most common causes of vitamin K deficiency in sepsis patients [16, 20, 21, 22, 23, 24].
We found no significant difference in PIVKA‐II levels and positive rate between benign obstructive jaundice and malignancy‐associated jaundice. Therefore, we believe that the elevation of PIVKA‐II is not due to carcinoma similar to HCC. Tartaglione et al. [15] hypothesized that the PIVKA‐II produced by pancreatic cancer was due to the common foregut derivation of the liver and pancreas, their embryologic proximity and their demonstrated capability of mutual trans‐differentiation. However, Kanazumi et al. divided patients with cholangiocarcinoma and pancreatic cancer without HCC into two groups. One group received 20 mg of vitamin K2 administered intravenously daily for 21 days after surgery, while the other group had no vitamin K2. PIVKA‐II levels of both groups decreased gradually to the normal range within about 2 weeks after the operation. In the group of patients without vitamin K2, PIVKA‐II levels increased 14 days after surgery [16], which confirmed our conclusion that the increase in PIVKA‐II levels in non‐hepatocellular carcinoma patients was not related to cancer.
Serum PIVKA‐II elevation remained common in these patients, even with vitamin K therapy. As demonstrated in this study, despite multivitamin supplementation in 24/58 patients with obstructive jaundice and 25/55 patients with sepsis, the association between PIVKA‐II and coagulation markers remained robust. Previous studies have reported that different vitamin K treatment regimens have varying effects on coagulation function [23]. This suggests that even with supplementation, intake is not sufficient to overcome the absorptive defect in these patients and current practices of oral vitamin K supplementation may be inadequate to maintain vitamin K nutriture in cholestatic liver disease.
To date, the underlying mechanisms responsible for the overexpression of PIVKA‐II in HCC have not been fully elucidated. Given the complexity of prothrombin production, the overexpression of PIVKA‐II in HCC tissues is likely driven by a combination of mechanisms. Current evidence indicates that elevated PIVKA‐II levels correlate with factors such as the hypoxic microenvironment, reduced activity of γ‐glutamyl carboxylase, impaired vitamin K metabolism, and overexpression of the prothrombin precursor [25, 26]. Furthermore, previous studies have demonstrated that PIVKA‐II promoted tumor growth, invasion and metastasis by enhancing cellular proliferation, extracellular matrix synthesis, and tumor angiogenesis [27].
In the presence of taking vitamin K antagonists or vitamin K deficiency, a special PIVKA‐II variant, known as NX‐DCP, can be abnormally elevated in non‐HCC subjects [28, 29]. However, NX‐DCP and HCC‐derived PIVKA‐II can be distinguished based on the number of glutamic acid (Glu) residues. PIVKA‐II derived from HCC patients typically carried 6–9 Glu residues, whereas NX‐DCP contained 1–5 Glu residues. Although routine tests using MU‐3 antibodies cannot distinguish differences in Glu residues, serum NX‐DCP levels can be detected by P‐11 or P‐16 antibodies. Nonetheless, the high variability between different examination batches and high cost limited the clinical application of P‐11 or P‐16 antibodies [30]. Nevertheless, several limitations should be taken into consideration. This was a single‐center retrospective study, which may be prone to selection bias. Additionally, among the 58 patients with obstructive jaundice, there were only eight cases of benign obstructive jaundice. The number of included cases was relatively small, which led to a certain margin of error.
In conclusion, the PIVKA‐II produced by HCC may differ from that produced by obstructive jaundice and sepsis. Despite multivitamin supplementation, elevated serum PIVKA‐II levels suggesting ongoing vitamin K deficiency are commonly observed in patients with obstructive jaundice and sepsis. Given the abnormally elevated serum PIVKA‐II levels in patients without hepatocellular carcinoma, these results should be interpreted with caution in patients with hepatocellular carcinoma combined with these diseases.

Ethics Statement
The study was approved by the Ethics Committee at Wuhan Union Hospital (the approved number 0887).

Consent
The authors have nothing to report.

Conflicts of Interest
The authors declare no conflicts of interest.

Supporting information

Table S1: jcla70128‐sup‐0001‐TableS1.docx.

Table S2: jcla70128‐sup‐0002‐TableS2.docx.

Table S3: jcla70128‐sup‐0003‐TableS3.docx.