Combination of hnRNP K, AFP and IL-22 as triple biomarkers predicts

the survival rate in liver cirrhosis patient for early detection and long term
prediction : A Novel Monitoring for Liver Cirrhosis Patient
Ilham Akbar R, Fadhilah Putri Wulandari, Siti Nurul Magfirah

Hasanuddin University, Makassar, Indonesia

1. Background

The word cirrhosis comes from the Greek word kirrhos, which means orange yellow (1). Laennec
gave cirrhosis its name kirrhos in 1819 in a brief footnote to his treatise De lauscultation mediate (2).
The definition of cirrhosis remains morphological, described by a working party for the World
Health Organization (WHO) in 1978 as: a diffuse process characterized by fibrosis and the
conversion of normal liver architectures into structurally abnormal nodules (3).

According to the World Health Organization (WHO), in 2006 approximately 170 million infected
human liver cirrhosis. This figure includes about 3% of the the entire human population in the world
and each year new infections cirrhosis hepatic increased 3-4 million people. The prevalence of disease
in the liver cirrhosis Indonesia, is not known. The prevalence of liver cirrhosis in 2007 in Indonesia
ranges from 1 to 2.4%. Of the average prevalence (1.7%), estimated at more than 7 million people in
Indonesia suffer from cirrhosis hepatic.

Certain reversible components of cirrhosis have been indicated where significant histological
improvement have occurred with regression of cirrhosis but complete resolution with a return to
normal architecture seems unlikely (5). The underlying immunological response has usually been
acting for months or years where inflammation and tissue repairing are in progress simultaneously
which leads in the end to fibrosis and cirrhosis (6).

Interleukin (IL)-22 is among newly identified parameters of hepatocyte biology that recently became
the major focus of basic and translational research on liver injury and inflammation [7]. This member
of the IL-10 cytokine family is primarily produced by activated CD4+ or CD8+ T cells, g-T cells,
macrophages/dendritic cells and a diverse array of natural killer (NK)-like cells recently coined innate
lymphoid cells [7-9]. IL-22 is biochemically and functionally akin to IL-6 and able to efficiently
initiate the hepatic acute phase response [10,11]. However, in contrast to IL-6, IL-22 almost
exclusively acts on non-leukocytic cells. As a result, cells of epithelial origin, including hepatocytes,
but not leukocytes, are major targets of IL-22 [7-9]. The potential of IL-22 as a parameter of liver
diseases is further highlighted by detection of its increased expression in patients liver biopsy
specimens using immunohistochemistry method [10,1213]. Enhanced levels of systemic IL-22 have
recently been observed in patients with chronic hepatitis [11] and acute hepatitis B infection by using
hematology and biochemically analysis in laboratorium [11].

The best currently evaluated prognosis score for patients with liver cirrhosis is the MELD score.
Systemic IL-22 levels in patients with liver cirrhosis significantly correlated with the MELD score,
substantiating that IL-22 is associated with deterioration of liver function and subsequent mortality of
cirrhotic patients. The MELD score is a short-term (three- to six-month) predictor of survival in
patients with end-stage liver disease, but is a weak predictor of survival in patients with compensated
liver cirrhosis in the long term. In multivariate analysis, systemic IL-22 was (independently from age,
presence of liver-related complications, elevated creatinine, high CRP and high MELD score)
associated with long-term mortality. Taking into account that IL-22 serum levels were stably
increased in the majority of patients in the course of liver cirrhosis
Hepatocellular carcinoma is one of the most common malignant tumors worldwide and is particularly
prevalent in China and Asia. Persisting viral infections such as Hepatitis B (HBV) and Hepatitis C
(HCV), which are the major common risk factors of HCC, is responsible for about 80% of all HCC
[14]. Chronic infection with HBV in the setting of cirrhosis increases the risk of HCC 70-fold [15].
The proteome of tumor tissue is a rich source of cancer biomarkers, and protein released from tumor
tissues may be more cancer specific than those from nontumor tissue. Investigation of the tumor tissue
proteome can identify proteomic signatures corresponding to clinicopathological features, and
individual protein in such signatures may be good biomarker candidate [16].

In this research, we investigate how the combination of Heterogeneous nuclear ribonucleoprotein K

(hnRNP K), AFP (Alfa feto protein) and IL-22 is the best monitoring method to do acute and long
term prediction in liver cirrhosis patient where hnRNP K-AFP will be used to detect early HCC and
IL-22 will be used to predict the long term condition to know other complications that might happen
in the future. This triple combination will help the doctor to detect early HCC and to know
complications disease in the future in liver cirrhosis patient so they can prevent it. In the end, we do
hope that this monitoring method will reduce the morbidity and mortality of liver cirrhosis patient by
doing early detection of HCC and long term prediction.

2. Material and Metods

This study uses systematic review method.

Literature searching

This study uses approved journal from many website.

Literature review

After journal and article have been collected, literature review was conducted to create a novel and
newest idea for monitoring method as the most needed research in liver cirrhosis disease as the
chronic hepatological disease.

3. Results

IL-22 serum levels are increased in patients with liver cirrhosis

To investigate whether liver cirrhosis is associated with increased serum IL-22, the cytokine was
determined in sera of healthy donors and liver cirrhosis patients, respectively. IL-22 was detectable
(>2.6 pg/ml) in 89 of 120 patients with liver cirrhosis, but only in 4 of 40 healthy controls (74.1% vs.
10.0%, P < 0.001) (Figure 1).. Age and gender were not associated with the systemic IL-22 level in
patients with liver cirrhosis (P > 0.2 for both). To determine reference range, we used the 95%
interval of IL-22 serum concentrations that were observed in healthy donors. Based on that strategy,
we defined the upper limit of normal (ULN) serum IL-22 concentration to be 18 pg/ml. According to
this ULN, 57 out of 120 (47.5%) patients with liver cirrhosis but only 2 out of 40 (5.0%) healthy
donors displayed elevated IL-22 serum levels.

IL-22 serum levels increase in the course of liver disease.

Next, we were interested whether IL-22 serum levels are stably increased in the course of liver
disease. Follow-up sera were available in 29 patients with liver cirrhosis. Thirteen patients (44.8%)
had elevated IL-22 serum levels at baseline. (P = 0.0192, Figure 2). Only 3 of 13 patients (23.1%)
with elevated IL-22 serum levels at baseline showed a decline of IL-22 below the ULN at followup
while 9 of 16 patients (56.3%) with IL-22 serum levels below the ULN at baseline showed an increase
of IL-22 above ULN at follow-up.

IL-22 is detectable in livers from patients with liver cirrhosis

A recent report demonstrates that IL-22 is produced locally in livers of patients with chronic viral
hepatitis [26]. In order to provide evidence that enhanced systemic IL-22 as observed herein likely
derived from diseased liver tissue, IL-22 expression was determined in liver biopsies by
immunohistochemical staining (available from only 10 patients, as liver biopsies are not routinely
performed in patients with advanced liver cirrhosis). IL-22 positive cells were observed in 7 of 10
liver biopsies from patients with different etiologies of liver cirrhosis. In agreement with Park et al.
[26], IL-22 expression was detectable mainly in non-parenchymal cells (Figure 3).

Serum IL-22 and etiologies of liver disease

We next investigated whether distinct etiologies of liver diseases in the patient cohort under
investigation affected IL-22 serum levels. Notably, no significant differences became apparent
between levels of IL-22 in sera from patients with liver cirrhosis due to chronic hepatitis B (HBV),
chronic hepatitis C (HCV) and alcoholic cirrhosis (AC) (P > 0.2). For hereditary, cholestatic,
autoimmune liver diseases as well as toxic liver injury and nonalcoholic steatohepatitis, the number of
patients was too low to draw a valid conclusion. Patients with chronic HBV, chronic HCV and
alcoholic cirrhosis had significantly higher IL-22 serum levels than healthy controls (P = 0.009, P <
0.001 and P < 0.001, respectively). These data do not support an association between the etiology of
the underlying liver disease and elevated serum IL-22.

Elevated IL-22 serum levels are associated with reduced survival of patients with liver cirrhosis
To investigate whether IL-22 serum levels are associated with survival of patients with liver cirrhosis,
we compared survival of patients with liver cirrhosis and normal IL-22 levels (below the ULN of 18
pg/ml) with survival of patients having elevated IL-22 serum levels (above the ULN of 18 pg/ml). As
illustrated in Figure 4, survival of patients with elevated IL-22 serum levels was significantly reduced
compared to patients with normal IL-22 serum levels (P = 0.003). The estimated mean survival time
was 526.4 days for patients with normal systemic IL-22 and 321.3 days for patients with elevated IL
22 (Figure 4).

IL-22 serum levels are associated with complications of liver cirrhosis

To investigate whether systemic IL-22 levels are associated with complications of liver cirrhosis, we
compared liver cirrhosis-related complications between patients with IL-22 serum levels above or
below the ULN of 18 pg/ml. Elevated IL-22 levels were more frequent in patients with liver cirrhosis-
related complications than in patients with compensated liver cirrhosis (60.0% vs. 17.1%, P < 0.001).
Moreover,elevated IL-22 serum levels were more frequent in patients with ascites, hepatorenal
syndrome (HRS) and spontaneous bacterial peritonitis as compared to patients without these
complications (Figure 5).

IL-22 serum levels correlate with MELD score

The currently best-evaluated prognostic score for patients with liver cirrhosis is the MELD score. In
the present study, there was a significant association between high MELD score (20) and reduced
survival (P = 0.017, hazard ratio (HR) 0.364, confidence interval (CI) (0.159 to 0.835)). As IL-22
serum levels were associated with mortality of patients with liver cirrhosis, we investigated the
relation between the MELD score and IL-22 serum levels. As shown in Figure 6, IL-22 serum levels
significantly correlated with the MELD score. The MELD score includes the laboratory parameters
for creatinine, bilirubin and international normalized ratio for prothrombin time (INR). Therefore, we
also investigated if the individual parameters of the MELD score correlate with serum IL-22 levels in
our patients. As illustrated in Table 1, creatinine and INR but not bilirubin correlated with IL-22
serum levels.

IL-22 serum levels correlate with surrogate parameters for inflammation

To investigate whether IL-22 serum levels are associated with determinants of liver synthetic
capacity, inflammation or damage, potential correlations of the cytokine with serum albumin
(surrogate marker of liver synthetic capacity), C-reactive protein (CRP, surrogate marker of ongoing
inflammation), and alanine aminotransferase (ALT) as well as aspartate aminotransferase (AST), both
surrogate markers of liver damage, were analyzed (Table 1). A strong positive correlation was found
between serum IL-22 and CRP levels (Table 1). Furthermore, weak but significant inverse
correlations between serum levels of IL-22 and albumin, as well as ALT, were observed (Table 1).
Overexpression of hnRNP K in early HCC tissue
Protein spots that showed at least two-fold changes and a significant difference in intensity (p<0.05)
between HCC and cirrhotic liver samples were included for further analysis. Among the proteins
identified as upregulated using mass spectrometry, the protein labeled SSP2215 was found to be
consistently overexpressed in HCC tissues compared with cirrhotic liver tissues (p<0.01) (Figure 7A).
This protein was expressed more strongly than other candidate biomarkers in all HCC tumors of
different stages (p<0.01) (Figure 7B). Because detection of early HCC in high risk subjects (e.g.,
those with cirrhosis and/or hepatitis B) could guide further treatment and improve patients clinical
outcomes, we were motivated to distinguish any potential biomarker with expression related to early
HCCs. Intriguingly, a stronger expression of SSP2215 was found to be significant in early HCCs
compared to other protein spots. Moreover, significant overexpression of this protein was maintained
in late HCC tumors, suggesting that expression of SSP2215 may be related to HCC development
(Figure 7C). Finally, the SSP2215 spot was identified as heterogeneous nuclear ribonucleoprotein K
(hnRNP K) by mass spectrometry .

HnRNP K is a potential biomarker for early HCC

To assess the individual performance of hnRNP K as a potential biomarker to discern early HCC from
cirrhosis, and to detect early HCC from late HCC in tissue, we selected optimal fixed cutoff
thresholds for hnRNP K and then calculated test sensitivity and specificity by receiver operating
characteristic (ROC) curves (Figure 8). At a cutoff threshold of 6.396 ppm, hnRNP K showed a high
accuracy to discern early HCC tissue from cirrhosis tissue with a sensitivity of 93.33% and a
specificity of 75% (AUC = 0.89, p<0.01) (Figure 8A). Likewise, hnRNP K separated early HCC from
late HCC at a cutoff threshold of 7.16 ppm with a sensitivity of 66.67% and a specificity of 84%
(AUC = 0.75, p<0.01) (Figure 8B). Serum AFP had a lower diagnostic capability (AUC = 0.60,
p>0.05) of detecting early HCC from late HCC either at a cutoff value of 100 ng/mL (Sen = 64.29%,
Spe = 56%) or 400 ng/mL (Sen = 64.29%, Spe = 40%) (Figure 8C), which is in accordance with the
acknowledged reports that AFP is insensitive for early HCC detection. It is well known that the
combination of multiple biomarkers will improves capability for disease diagnosis. We used fixed
cutoff thresholds of 7.16 ppm hnRNP K and 100 ng/mL AFP to discern early HCC from late HCC
with a sensitivity of 93.33%, specificity of 44% and accuracy of 62.5%. In contrast, the serial test,
with fixed cutoff thresholds of 7.16 ppm hnRNP K and 100 ng/mL AFP, requires that both hnRNP K
and AFP are abnormal, and it increased specificity and accuracy to 96% and 72.5%, respectively, at
the expense of sensitivity (33.33%) when early HCC were compared to late HCC. If a higher
threshold of AFP (400 ng/mL) was combined with hnRNP K, the overall accuracy decrease either in
the parallel test (62.5% vs 55%) or in the serial test (72.5% vs 70%), suggested that the combination
of fixed hnRNP K with relative lower AFP (100 ng/mL) as a cutoff threshold was more powerful to
diagnose early HCC.

4. Discussion

The pathogenesis of liver cirrhosis from all three etiologies which are HBV, HCV, and
Alcoholic liver cirrhosis consists of 4 stage: 1. Redox alterations, 2. Oxidant stresses, 3. Inflammatory
cell infiltration and activation, and 4.Centrilobular hypoxia. First, ADH mediated Etholoxidation leads
to reduction of oxidized (NAD+) to NADH. Increased NADH shifts redoxstate of hepatocytes which
affects other NAD+ dependent processes including Lipid & CHO metabolism leading to hepatic
steatosis which NADH provoke steatosis by stimulating fatty acid synthesis & inhibiting
mitochondrial beta oxidation. Fatty acids accumulate in hepatocytes& are stored as TGs. Second,
Ethol oxidant leads to formation of free radical species hydroxyethyl, super oxide, & hydroxyl
radical which inflicts oxidative damage to intracellular compounds which consists 4 process: 1. attack
unsaturated lipids causes lipid peroxidation and lead to tissue damage & fibrosis, 2. attack DNA
causing deletion & mutations and lead to mitochondrial dysfunction, 3. decrease antioxidant defenses
by decreasing amounts of Vitamin A and E which increase hepatic lipid peroxidation and cause
lysosomal damage, and 4. decrease glutathione. Third, induce 2 process, 1.Kuppfer cell activation &
cytokine production example TNF, IL 1, IL 6, IL 8 causing oxidative injury, 2. Immune response to
altered hepatocellularproteins (caused by oxidative injury) leading to formation of Abs. And the last,
fourth, due to increase O2 demand for ethanol metabolism, a zone of hypoxia around central veins,
which is the farthest from oxygenated blood, develops. (Figure 10)

The potential role of IL-22 in liver diseases has been intensively studied in murine models for T cell-
mediated hepatitis [24], fulminant hepatic failure [30], alcoholic liver injury [22] and regeneration
after hepatectomy [25]. In those models, IL-22 attenuated liver injury [20,22], prevented hepatic
failure [24] and improved hepatic steatosis [24]. On the other hand, blockage of IL-22 bioactivity
increased liver injury [20] and was associated with decreased hepatocyte proliferation following
hepatectomy [31]. On the whole, with the exception of experimental hepatitis B virus infection [32],
murine models largely suggest a tissue protective function of IL-22 in hepatic disorders.

IL-22 sera contents in healthy donors were, for the most part, barely detectable and set the basis for
calculation of a reference range. This reference range defined levels below 18 pg/ml as being normal
(ULN), which agrees with previous reports on IL-22 in sera of healthy donors obtained in the US and
Europe [35, 36, 37].

According to this threshold, 47.5% of patients with liver cirrhosis showed elevated IL-22 serum
concentrations. Follow-up analyses of serum IL-22 levels in patients with liver cirrhosis suggest that
mean IL-22 levels increase during the course of liver disease. The majority of patients with elevated
IL-22 baseline levels maintain elevated levels during follow-up, while more than half of patients with
normal IL-22 serum levels at baseline develop increased levels during follow-up. These results
indicate that IL-22 elevation is not a transient phenomenon in patients with liver cirrhosis. The
mechanisms mediating this IL-22 increase in the patients are not yet clear. However, it can be
assumed that increasing IL-22 levels are connected with increased cytokine production as well as
reduced hepatic or renal elimination. No difference between IL-22 produced in cirrhosis liver by
different etiologies.

The MELD score includes three blood surrogate parameters addressing different aspects of liver
deterioration. INR and bilirubin reflect liver synthetic capacity and excretory function, while
creatinine indicates renal decompensation due to hepatic failure. IL-22 serum levels correlated with
two parameters of the MELD score, that is, creatinine and INR. Furthermore, weak inverse
correlations were observed between systemic IL-22 and serum albumin and ALT. IL-22 also
correlated with CRP, a well-established surrogate marker of hepatic inflammation and prognosis of
liver cirrhosis. CRP and creatinine were both associated with serum IL-22, further suggesting that
enhanced systemic IL-22 is driven by hepatic inflammation along with renal deterioration. Whether
IL-22 bioactivity likewise contributes to renal deterioration is unknown.

We identify disease-related proteins present in the early HCC tumor tissues by tissue proteomics,
which would lead to a better understanding of the mechanisms driving tumor development could
provide useful biomarkers for early detection and prognostic prediction. HnRNP K was selected to be
identified and further validated both because of its high expression level in HCC tissue compared to
the cirrhosis control, and its capability of distinguishing early HCC from late HCC. Validation of
aberrant expression of hnRNP K in additional independent HCC tissues further reinforced the use of
hnRNP K as a potential tumor marker. Correlation analysis showed that hnRNP K was not only a
potential biomarker for the detection of early HCC, but also the expression level of this protein was
positively correlated with the increased tumor size and the presence of microsatellites. This
demonstrated that hnRNP K overexpression may be related to active tumor growth and intrahepatic
micrometastasis.

In the present study, we found that hnRNP K is overexpressed in individuals with HCC of all sizes
and that it could distinguish early HCC from late HCC. This makes it a candidate biomarker for HCC
screening in patients with high risk HBV infection. We compared the capability of tissue hnRNP K
with serum AFP (cut off thresholds: 100 ng/mL) in diagnosing early HCC, and as expected, hnRNP K
showed a better performance than AFP in detecting early HCC (sensitivity: 66.7% vs. 64.29%,
specificity: 84% vs. 56%). Because a single biomarker will not provide information regarding both
tissue type and malignant transformation throughout the various stages of tumor development and
progression, we further combined tissue hnRNP K intensity and serum AFP concentration to form a
biomarker panel. This enhanced both the sensitivity and specificity by parallel and serial tests.The
results showed that parallel test with tissue hnRNP K intensity and serum AFP cutoff thresholds
optimized sensitivity (93.33%), whereas serial test optimized test specificity (96%). The combined
use of hnRNP K and serum AFP has improved utility for screening and diagnosing early HCC in
cirrhotic tissue. Our study describes for the first time the usefulness of hnRNP K as a tumor
biomarker for detecting early HCC, especially the detection of early HCC from liver cirrhosis. The 2-
DE and immunohistochemistry data showed that hnRNP K is a specific biomarker for tumor tissue.
Detecting hnRNP K expression in tissue may facilitate the accuracy of HCC diagnosis. Both the
general histodiagnosis of small nodules and the distinction of high-grade dysplastic nodules form
early HCC are extremely challenging, a positivity of hnRNP K staining in tissue could be taken as
indicator of HCC. (Figure 9)

5. Conclusion

Elevation of IL-22 levels are predictive for reduced survival in patients with liver cirrhosis
independent of age, presence of liver related complications, CRP, creatinine and the MELD score.
Our data indicate that processes in the liver that lead to deterioration of liver cirrhosis and its sequelae
are associated with an increase of IL-22. In here, we demonstrate cut-off threshold for IL-22 is > 18
pg/ml. To diagnose fastly present of tumor tissue type and malignant because a single biomarker will
not provide information regarding both tissue type and malignant transformation throughout the
various stages of tumor development and progression, we further combined tissue hnRNP K intensity
and serum AFP concentration to form a biomarker panel. This enhanced both the sensitivity and
specificity by parallel and serial tests. The results showed that parallel test with tissue hnRNP K
intensity and serum AFP with cut-off threshold hnRNP K 7.160 ppm and AFP 100 ng/mL
optimized sensitivity (93.33%), whereas serial test optimized test specificity (96%). The combined
use of hnRNP K and serum AFP has improved utility for screening and diagnosing early HCC in
cirrhotic tissue. The combination between IL-22 > 18 pg/ml and heterogeneous ribonucleoprotein K
(hRNP K)-AFP 7.160 ppm and 100 ng/mL respectively will bring a better prognosis and
prediction, early detection and long term prediction, respectively. (Table 2)

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