Role of Myeloperoxidase in hepatitis C virus related hepatocellular carcinoma

Accepted Manuscript Role of myeloperoxidase hepatocellular carcinoma

in

hepatitis

C

virus

related

Mohamed Abdel-Hamid, Ola Hassan Nada, Doha El-Sayed Ellakwa, LamiaaKhalaf Ahmed PII: DOI: Reference:

S2214-5400(18)30153-1 doi:10.1016/j.mgene.2018.07.008 MGENE 472

To appear in:

Meta Gene

Received date: Revised date: Accepted date:

1 May 2018 12 July 2018 17 July 2018

Please cite this article as: Mohamed Abdel-Hamid, Ola Hassan Nada, Doha El-Sayed Ellakwa, LamiaaKhalaf Ahmed , Role of myeloperoxidase in hepatitis C virus related hepatocellular carcinoma. Mgene (2018), doi:10.1016/j.mgene.2018.07.008

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ACCEPTED MANUSCRIPT Role of Myeloperoxidase in Hepatitis C Virus related Hepatocellular carcinoma Mohamed Abdel-Hamid1, Ola Hassan Nada2, Doha El-Sayed Ellakwa3, LamiaaKhalaf Ahmed 3 Microbiology Department, Faculty of Medicine, Minia university

2

Pathology Department, Faculty of Medicine, Ain Shams University

3

Biochemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University

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1

ACCEPTED MANUSCRIPT Abstract Introduction: Hepatitis C infection has the ability to create substantial oxidative stress inside hepatocytes. In this way, oxidative stress has been distinguished as a significant mechanistic pathway culminating in the improvement of hepatic cirrhosis, liver failure and liver cancer. Aim of

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this study: is to detect the probable role of immunohistochemical expression of Myeloperoxidase in the improvement of HCV associated

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HCC and also, liver cirrhosis and to correlate the expression of MPO with

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different clinico-pathological features of HCC. Subjects and Methods: This study was conducted on a total number of 59patients. These were

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subdivided into 25patients of hepatocellular carcinoma and 34 patients of chronic liver diseases (CLDs) with cirrhosis. All studied 59patients were with

HCV

immunohistochemically

to

Results:

All

patients

were

examined

demonstrate

the

expression

of

Concerning

the

expression

of

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Myeloperoxidase.

infection.

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associated

Myeloperoxidase in HCC and cirrhoticpatients; the study revealed that

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more MPO expression was found in HCC cases when compared with cirrhoticpatients (P –value <0.0001). conclusion, the study established a significantly higher expression of Myeloperoxidase in HCV related HCC

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compared to cirrhosis. This clarifies a crucial role of Myeloperoxidase in the process of HCV induced hepatocarcinogenesis. Key words: Myeloperoxidase , liver cirrhosis, Hepatocellular carcinoma , oxidative stress .

ACCEPTED MANUSCRIPT 1. Introduction Liver disease is a major cause of morbidity and mortality here in Egypt. Hepatitis C virus infection (HCV) is the main cause of liver disease, with 70,000 up to 140,000 newly registered cases each year. Egypt maintains first position in the occurrence of HCV globally (Gomaa et al., 2017).

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The genotype 4a is the major common genotype in country (AbdelHamid et al., 2015). Cirrhosis is the most threatening risk factor for HCC

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giving little respect to etiology and could be caused by chronic viral primary

biliary

cirrhosis,

and

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hepatitis (mainly Hepatitis B Virus and Hepatitis C Virus), Stage 4 metabolic

diseases

like

genetic

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hemochromatosis, and non-alcoholic fatty liver disease (Flores and Marrero, 2014). Oxidative stress plays an important role in the

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pathogenesis of cirrhosis and its complications, Together with cytokinemediate apoptosis, reactive Oxidative stress (ROS) could cause cell death

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and the possible development of liver failure. ROS could also cause DNA damage, somatic mutations, and the possible development of a HCC

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(Magdaleno et al., 2017). Myeloperoxidase is an important enzyme involved in the generation of reactive oxygen species. In the presence of physiological chloride concentrations, MPO reacts with hydrogen

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peroxide (H2O2, formed by the respiratory burst) to catalyze formation of hypochlorous acid/hypochlorite (HOCl/OCl−) and other oxidizing species (Ho et al., 2013). These oxidants may participate in damaging the host tissue at sites of inflammation via reactions with many of biological substrates, which include DNA, lipids, and protein amino groups (Do Carmo et al., 2014). MPO-H2O2 framework can additionally form reactive nitrogen species in the absence of physiological chloride concentrations that might begin lipid peroxidation or form protein tyrosine residues, an additional posttranslational adjustment existed

ACCEPTED MANUSCRIPT previously in lots of pathological cases (Samuel et al., 2015). The presented study was performed to investigate the probable role of immunohistochemical expression of Myeloperoxidase in the development of HCV associated HCC as well as liver cirrhosis. It is worth mentioning that none of the immunohistochemical reports focused on the role of

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Myeloperoxidase in HCV-associated HCC. 2. Subjects and Method

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The study was conducted on a total number of 59 patients. These were

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subdivided into 25 subjects suffering from hepatocellular carcinoma and 34 subjects with chronic hepatitis C (CHC) with cirrhosis. All studied 59

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subjects were associated with HCV infection. Samples were collected as paraffin embedded tissue blocks from Viral Hepatitis Research Lab

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(VHRL) located in the National Hepatology and Tropical Medicine Institute (NHTMI), Cairo. Written, informed consent was obtained from

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each patient before enrollment into the study. The sera of patients were collected for liver function tests (AST & ALT), AFP test, HCV antibody,

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and HCV RNA and HBs Ag tests. Histopathologic study was carried out by examining the Hx&E slides for confirmation of the diagnosis of HCC and CLD subjects and grading of the tumors. Immunohistochemical

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staining. Immunohistochemical staining of the formalin fixed, paraffin embedded tissue sections (4µm thick) was performed following the technique of Hsu et al, (1981). Briefly, the tissue sections were deparaffinized and rehydrated in xylene and descending grades of alcohol, rinsed in phosphate-buffered saline (PBS, PH 7.6). Antigen retrieval was done for 10 min in EDTA solution (PH 8), using a microwave oven (700W). After cooling down at room temperature, endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Then, the tissue sections were incubated overnight with Rabbit

ACCEPTED MANUSCRIPT polyclonal antibodies against human Myeloperoxidase (Dako Inc., Denmark Code IS511). Each antibody was supplied in a concentrated form and diluted 1:100 in PBS. The antibodies were detected with the avidin–biotin detection kit (Lab vision, USA, Catalog number: TP-060HL)

using

diaminobenzidine

(DAB)

as

the

chromogen.

After

counterstaining with haematoxylin, the tissue sections were washed with

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distilled water, dehydrated with ascending grades of alcohol and xylene

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and finally mounted with mounting medium. Lymphocytes and stromal cells adjacent to or admixed in the tissue were considered as internal

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positive control. For each case, a tissue section following the same steps antibody,

served

as

the

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of immunohistochemical technique, except for omission of the primary negative

control.

Interpretation

of

control

were

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immunostaining: Only cases with appropriate staining of the positive considered

suitable

for

evaluation.

The

immunohistochemical expression of MPO in liver biopsies was examined

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in malignant hepatocytes of HCC and in each of Kuppfer cells, polymorphonuclear leucocytes, hepatocytes and fibrous tissue septa of

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cirrhotic tissue and was graded as: Negative (-) i.e.: negatively stained cells (no brown staining). Mild (+) i.e.: positively stained cells are <15

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cells/40magnification). Moderate (++) i.e.: positively stained cells are 1530 cells/40 magnification). Marked (+++) i.e.: positively stained cells are >30 cells/40 magnification) per section (Bekheet et al., 2009).

Statistical Analysis: Student T Test: was used to assess the statistical significance of the difference between two study group means. Chi-Square test: was used to examine the relationship between two qualitative variables. The one-way

ACCEPTED MANUSCRIPT ANOVA test: for testing statistical significant difference between means of more than two groups. Correlation analysis: The correlation coefficient denoted symbolically r, defines the strength and direction of the linear relationship between two variables. Spearman correlation coefficient (non- parametric test): to test the association between two or more P< 0.05: Significant (S), P<0.01: Highly significant.

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Results

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variables.P- value: level of significance; P>0.05: Non significant (NS),

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HCC patients: the patients' age ranged between 47 and 75 years with a mean age of 60.12 years (± 1.799). Nineteen patients were males (76%)

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and the remaining six patients were females (24%) with the male: female ratio of 3.1: 1. And the ages of the patients with CLDs ranged between 42

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and 70 years with a mean age of 55.79 years (± 1.344). Twenty eight patients were males (82.35%) and six patients were females (17.65%)

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with the male: female ratio of 4.6:1as shown in table (1).

HCC (n=25)

Cirrhosis (n=34)

(47-75) 60.12 ± 1.799

(42-70) 55.79 ± 1.344

19(76%) 6 (24%)

28 (82.35%) 6 (17.65%)

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Age (years) Range Mean ±SEM Gender Male: (%) Female: (%)

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Table 1 Age and Gender distribution among studied patients.

Results of Laboratory findings : The data showed that all HCC and cirrhotic cases were seropositive for HCV antibody and HCV RNA and seronegative for HBV surface antigen.

ACCEPTED MANUSCRIPT The mean values of serum AFP level were highly significant different in the studied groups as it was more in HCC group when compared to LC group (P -value < 0.0001).It was found that 68% of HCC patients, 100% of cirrhotic patients had AFP serum levels < 200 ng/ml, in addition, when mean values of serum level of AFP < 200 ng/ml were compared between studied groups, there was highly significant difference between HCC and

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LC groups (P -value < 0.0001),On the other hand, 32% of HCC patients

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had AFP serum levels > 200 ng/ml. The mean values of serum level of AFP > 200 ng/ml when compared between the studied groups, there was

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highly significant difference between HCC and LC groups (P -value <

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0.0001) as shown in table (2).

Table 2 AFP serum levels in the studied groups.

AFP Range Median Mean ±SEM

94-410 170 178.5± 16.58

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AFP < 200 ng/ml N (%) Mean ±SEM

AFP > 200 ng/ml N (%) Mean ±SEM

LC n=34

P-value

1-10 4 4.779 0.5534

< 0.0001

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HCC n=25

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Serum AFP level

±

17 (68%) 34 (100%) < 0.0001 134.4 ± 4.779 ± 8.976 0.5534 8 (32%) 272.3± 26.59

0 (0%) 0

< 0.0001

Alpha-fetoprotein serum level was done in all 25 HCC cases. In these patients it ranged between 94 and 410 ng/ml with a mean level of 178.5 ± 16.58 ng/ml , AFP serum level was <200 ng/ml (cut off level) in 17 HCC

ACCEPTED MANUSCRIPT cases (68%), while 8 HCC cases (32%) had serum AFP level ≥ 200 ng/ml b) Liver function tests : Liver biochemical profile of the studied cases were done and showed that, the mean value of AST level was 61.88 ± 5.790 and 57.32 ± 4.271 among the HCC cases and cirrhotic cases respectively. And the mean value of

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ALT level was 64.20 ± 6.495 and 60.71 ± 5.053 among the HCC cases

non-significant(p >0.05) as shown in table (4).

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and cirrhotic cases respectively. However these results were statistically

Mean ±SEM 61.88 ± 5.790

ALT

64.20 ± 6.495

P-value

Mean ±SEM 57.32 ± 4.271

0.5195

60.71 ± 5.053

0.6681

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AST

LC (N=34)

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Parameter HCC (N=25)

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Table 3 Mean values of AST and ALT among studied cases.

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P > 0.05: Non significant (NS), P ≤ 0.05: Significant (S).

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Results of immunohistochemical examination: A positive brown cytoplasmic immunohistochemical staining was

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identified in 22(88) % out of 25 cases of HCC ,while 3 (12) % showed negative expression figure (1) .The positive immunohistochemical staining cases were further subdivided in to 15 cases representing 68.2 % with marked expression of MPO figure (2) and 7 cases 31.8 % showed moderate expression of MPO figure (3). None of the positive HCC cases showed mild expression. Regarding studied cases with liver cirrhosis, Myeloperoxidase positive immunostaining was encountered at different sites and in each; we scored

ACCEPTED MANUSCRIPT the expression and compared these findings with demographic data,

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laboratory findings and MPO expression in HCC cases figure (4).

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Fig. 1 A case of HCC showing Fig. 2 A case of HCC (trabecular negative immunostaning of variant) showing marked immunoMyeloperoxidase (IPx 400 staining of Myeloperoxidase (IPx 200

Fig. 3 A case of HCC (trabecular variant) showing moderate immuno -staining of Myeloperoxidase (IPx 200).

Fig. 4 A case of cirrhosis showing positive immunostaining of Myeloperoxidase in hepatocytes and kupffer cells (IPx 200).

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Kupffer cells showed the highest positive MPO expression 28 (82.4%) compared to other immuno-stained locations in the tissues showing cirrhosis. On the other hand PNL in sinusoids showed the highest percentage of negative expression 31(91.2%).This inverse relationship was highly statistically significant (P <0.0001) As shown in table (5) and

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Fig 5.

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Table 4 Myeloperoxidase tissue expression in various locations in

MPO tissue expression

+ve expression No (%) 28(82.4) %

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Kupffer cell

-ve expression No (%) 6 (17.6) %

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Liver cirrhotic Cell

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cirrhotic cases.

3(8.8)%

Hepatocyts

13(38.2)%

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PNL in 31(91.2)% sinusoids Fibrous 10(29.4)% tissue septa

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21(61.8)%

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χ 2 = 44.94

24(70.6)%

significant

P value < 0.0001

ACCEPTED MANUSCRIPT

MPO expression

80

-ve +ve

P- value<0.0001 60 40 20 0 cirrhotic hepatocytes

septa

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PNL

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kupffer

Fig 5 Myeloperoxidase tissue expression in various tissues in cirrhotic

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SC

cases.

B- Relation between MPO expression status and gender of the studied

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cases:

It was found that 17 (68 %) of HCC subjects with male gender showed

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positive expression while the remaining 2 (8 %) showed negative expression. Meanwhile 5 (20 %) of HCC subjects with female gender

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showed positive expression of MPO while 1 (4 %) showed negative expression. No statistically significant relationship was found between

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MPO expression and the gender of HCC patients as shown in table (6).Also it was found that all females of cirrhotic subjects had positive MPO tissue expression Meanwhile males of cirrhotic subjects had both positive and negative MPO expressions. This difference was not statistically significant as shown in table (7).

ACCEPTED MANUSCRIPT Table 5 Relation between Myeloperoxidase expression and the gender of the HCC patients. MPO

tissue

expression patients

features

-ve

+ve

No.

No. (%)

(%) Total number

25

3(12)%

22(88)%

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Gender 19(76)% 2(8)%

Female

6(24)%

1(4)%

17(68)% 5(20)%

P- value = 0.6866

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χ 2 = 0.1628

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Male

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of

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No

pathological

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Clinic-

ACCEPTED MANUSCRIPT Table 6 Relation between Myeloperoxidase expression and the gender of cirrhotic subjects. MPO tissue expression patients

Total

34

-ve

+ve

No. (%)

No. (%)

6

(17.6 28 (82.4 %)

%) 28

(82 6 (18 %) 22 (64 %)

%)

Female

0 (0%)

P- value=0.2115

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χ2= 1.561

6 (18%)

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6 (18%)

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Male

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number

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of

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No.

Gender

C-Relation between MPO tissue expression and biochemical parameters

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in studied subjects:

Marked MPO tissue expression was associated with subjects that had

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AFP >200 ng/ml while subjects with < 200 ng/ml had positive and negative MPO expressions. This distribution was not statistically significant (P-value=0.2053) as shown in table (8).No statistically significant relationship was found between MPO expression and each of ALT and AST of HCC subjects as shown in table (9).No statistically significant relationship was found between MPO expression and each of ALT and AST in cirrhotic subjects as shown in table (10).

ACCEPTED MANUSCRIPT Table 7 Relationship between Myeloperoxidase tissue expression and serum AFP level in HCC Patients. Serum AFP

MPO tissue expression

Total

-ve

+ve

No. (%)

No. (%)

<200 ng/ml

3 (12 %)

14 (56%)

>200 ng/ml

0 (0 %)

8 (32%)

`Total

3 (12 %)

22 (88 %)

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8 (32 %)

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P- value=0.2053

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χ 2 = 1.604

17 (68 %)

25 (100 %)

ACCEPTED MANUSCRIPT Table 8 Relation between Myeloperoxidase expression and biochemical parameters in HCC subjects. Parameter

HCC subjects

P- value

-ve

Moderate

Marked

No.= 3

No.= 7

No.= 15

Range

(30-44)

34-108

28-150

Median

36

60

57

60

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0.1240

66

80.57± 18.72 62.27 ± 5.439

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Mean ± SEM 35.67± 0.333

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36

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Median

40-180

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35-36

30-110

60.71± 9.588 67.47± 8.098

ALT (U/L) Range

0.2496

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Mean ± SEM 36.67± 4.055

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AST (U/L)

ACCEPTED MANUSCRIPT Table 9 Relation between Myeloperoxidase expression and biochemical parameters in cirrhotic subjects. Parameter

Liver cirrhosis subjects (kupffer cell) Mild

Moderate

Marked

No.=12

No.=13

No.=3

Range

(30-150)

28-81

44-64

Median

71.5

44

50

0.0829

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AST (U/L)

P- value

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Mean ± SEM 68.75 ± 9.061 46.69 ± 4.374 52.67± 5.925

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ALT (U/L) 34-110

30-79

36-96

Median

67.5

46

46

0.1321

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Range

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Mean ± SEM 66.25 ± 6.226 49.08 ± 4.633 59.33 ± 18.56

studied cases:

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Relation between Expression of Myeloperoxidase and histopathology of

I-Expression of Myeloperoxidase in Kupffer cells of cirrhotic cases:

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A statistically significant distribution was obtained when comparing the degree of MPO expression in HCC and kupffer cells of cirrhotic subjects. More marked expression of MPO was found in HCC subjects when compared with the expression of MPO in kupffer cell of cirrhotic subjects with P-value < 0.0001 as shown in table (11).

ACCEPTED MANUSCRIPT Table 10 Degree of expression of Myeloperoxidase in kupffer cell of cirrhotic subjects compared to HCC subjects. MPO tissue expression

No. (%)

No. (%)

Moderate(+ Marked(+++ +) ) No. (%)

LC(kupff er)

6 (10 12 %) %)

(20.5 13 (22 %)

HCC

3 (5 %)

Total

9 (15 12 (20 %) 20 (34 %) %)

7 (12 %)

3 (5 %)

15 (25.5 %)

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0 (0 %)

No. (%)

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Mild (+)

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Negativ e

Total

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P- value< 0.0001

25 (42.5%) 59 (100 %)

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χ 2= 21.94

18 (31 %)

34 (57.5 %)

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II-Expression of MPO in PNL in sinusoids of cirrhotic cases: We found that by comparing the degree of immunohistochemical

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expression of MPO in PNL in sinusoids of cirrhotic subjects with that in HCC subjects, marked expression was only detected in HCC subjects and

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most of PNL in sinusoids of cirrhotic subjects showed negative MPO expression when compared to HCC immunostaining. This inverse relationship was also highly statistically significant (P < 0.0001) as shown in Table (12).

ACCEPTED MANUSCRIPT Table 11 Comparison between degree of expression of Myeloperoxidase in (PNL) in sinusoids of cirrhotic and HCC subjects. MPO tissue expression

Total

Mild (+)

Moderate(++)

Marked(+++)

No (%)

No (%)

No (%)

No (%)

31 (52.5)%

1(2%)

2(3)%

0(0)%

HCC

3(5)%

0(0)%

7(12)%

Total

34(57.5)%

1(2)%

9(15)%

LC

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Negative

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χ 2 = 41.43

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(PNL)

15(25.5)%

25(42.5)%

15(25.5)%

59(100)%

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P< 0.0001

34(57.5)%

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III-Expression of MPO in fibrous tissue septa of cirrhotic cases: HCC subjects had more MPO expression when compared with MPO expression in fibrous tissue septa of LC subjects, and none of HCC

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subjects showed mild degree of MPO expression in comparison to cirrhosis that had mild immunohistochemical expression in fibrous tissue

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septa in 9 out of 34 subjects, these findings were highly statistically

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significant (P=0.0031) as shown in table (13).

ACCEPTED MANUSCRIPT Table 12 Degree of expression of Myeloperoxidase in fibrous tissue septa of LC subjects compared to HCC subjects. MPO tissue expression -ve No (%)

Mild (+)

Total

Moderate(++) Marked(+++) No (%)

No (%)

HCC

3 (5%)

0 (0 7 (12 %) %)

Total

13(22%) 9 (15 14 (24 %) %)

15 (25.5 %)

25(42.5%)

23 (39 %)

59(100 %)

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P= 0.0031

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χ 2= 13.85

34(57.5%)

8 (13.5 %)

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9 (15 7 (12 %) %)

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LC in fibrous 10 tissue septa (17%)

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No (%)

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IV-Expression of MPO in hepatocytes of cirrhotic cases : HCC subjects showed marked MPO expression when compared

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with MPO expression in hepatocytes of LC subjects that mostly showed mild degree of expression, while no mild expression of MPO was detected in HCC subjects, these findings was highly statistically

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significant ( P <0.0001) as shown in table (14).

ACCEPTED MANUSCRIPT Table 13 Degree of expression of Myeloperoxidase in hepatocytes of cirrhotic subjects compared to HCC subjects. MPO tissue expression -ve No (%)

Mild (+)

Total

Moderate(++) Marked(+++) No (%)

No (%)

3 (5%)

0 (0 7 (12 %) %)

Total

13(22%)

9 (15 14 (24 %) %)

15 (25.5 %)

25(42.5%)

23 (39 %)

59(100 %)

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P <0.0001

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HCC

χ 2= 29.08

34(57.5%)

2 (3.5 %)

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LC in 21(35.5%) 6 (10 5 (8.5 %) hepatocyts %)

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No (%)

The previously mentioned results showed that Myeloperoxidase

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expression was significantly higher in HCC cases compared to cirrhotic

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cases. Discussion:

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Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancerrelated death worldwide and one of the leading causes of death in patients with cirrhosis. HCC is projected to surpass breast and colorectal cancer to become the 3rd leading cause of cancer-related death in the United States by 2030 (Abdel-Hamid et al., 2014). Currently only 46% of HCC patients are diagnosed at an early stage and most do not receive curative therapy (Singal and El-Serag, 2015). Liver carcinogenesis is a complex and multifactorial process, in which both environmental and genetic features interfere and contribute to malignant transformation. Patients with cirrhosis are particularly exposed and justify periodical screenings in

ACCEPTED MANUSCRIPT order to detect the early development of hepatocellular carcinoma. The risk of HCC is, however, not identical from one patient to another. The identification of host factors that may also play an important role in HCC development may improve our understanding of the implications of the various biological pathways involved in liver carcinogenesis (Nahon and Rossi, 2012). Hepatitis C virus (HCV) is one of the major etiologic agents

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that causes HCC by generating an inflammatory, fibrogenic and

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carcinogenic tissue microenvironment in the liver, chronic infection with HCV has been responsible for the increasing HCC incidence in developed

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countries (Hoshida et al., 2014). Egypt Health Issues Survey (EHIS),

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2015 reported that 6 % of individuals' age 1-59 years old had a positive result on the hepatitis C antibody test. About 4% of them, or an estimated

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3.5 million Egyptians, were found to have an active infection (Demographic health system, 2015).

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Hepatitis C virus has the capacity to generate substantial oxidative stress within hepatocytes. Subsequently, oxidative stress has been

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identified as a significant mechanistic pathway culminating in the development of hepatic cirrhosis, liver failure and liver cancer (Lozano-Sepulveda et al., 2015). The host immune response to HCV

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infected hepatocytes is responsible for liver injury and subsequent development of hepatic fibrosis. The cellular elements comprising the host immune response include neutrophils, macrophages (Kupffer cells) and lymphocytes. Neutrophils and Kupffer cells contain myeloperoxidase (MPO) (Odobasic et al., 2016). Myeloperoxidase is an oxidative enzyme released from stimulated polymorph nuclear leukocytes and phagocytes at sites of inflammation. It has been involved in the pathogenesis of several diseases through

ACCEPTED MANUSCRIPT excessive production of reactive oxygen species (ROS) as well as through its genetic polymorphism (Ali et al., 2009). Inflammation activates a variety of inflammatory cells, which trigger oxidant-generating enzymes such as inducible nitric oxide synthase (iNOS), NADPH oxidase, and myeloperoxidase to produce high

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concentrations of free radicals including reactive nitrogen species (RNS) and reactive oxygen species. Overproduction of RNS and ROS can

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change the balance of oxidants and antioxidants and cause oxidative

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stress which contributes to the damage of biomolecules such as DNA, RNA, lipid and proteins, leading to an increase in mutations, genomic

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instability, epigenetic changes, and protein dysfunction and play roles in the multistage carcinogenic process (Murata et al., 2012).

immunohisto-chemical

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The present study was aimed to detect the probable role of expression

of

Myeloperoxidase

in

the

ED

improvement of HCV associated HCC and also, liver cirrhosis and to correlate the expression of MPO with different clinico-pathological

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features of HCC. It is worth mentioning that, it is the first study to provide novel information about the role of myeloperoxidase in HCV

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related HCC using immunohistochemistry in Egypt. The current study was conducted on a total number of 59patients. These were subdivided into 25patients suffering from hepatocellular carcinoma and 34 patients with chronic hepatitis C (CHC) with cirrhosis. All studied 59patients were associated with HCV infection. In the present study, the mean age of HCC patients was (60.12 ± 1.799) years i.e., the Sixth decade of life. These findings are in accordance with the study done by Abdel Wahab et al., (2007) who noted that the mean age of HCC patients was (54.26 ± 9.2) years. On the other

ACCEPTED MANUSCRIPT hand, a study done by El-Zayadi et al., (2005), revealed a slight shift of HCC incidence to younger age group. This controversy may be explained by the occurrence of HCV related HCC in an older age group in the present study and small sample size. Regarding the gender of HCC patients, the present study showed a

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male predominance with a male to female ratio 3.5:1. These finding

remarkable male predilection in HCC patients.

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agreed with study done by Do Carmo et al., (2012) who found a

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An increased carrier rate of hepatitis among males than females can be explained by the higher expression of androgen receptors in HCC

MA

predisposition (Hussein, 2004).

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rather than the expression of estrogen receptors and the possible genetic

In this work, the expression of MPO was correlated with the gender of all the studied patients, and non-statistically significant

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relationship between expressions of MPO and the gender of HCC and LC

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patients was found. This finding coincides with that reported by Do Carmo et al., (2012).

Also Kumar et al., (2004)found no direct association between sex

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and MPO levels. This was explained by the higher age of women in their study, since MPO level was shown to be influenced by the estrogen. This was incompatiblewith study done by Do carmo et al., (2014)who reported that, MPO polymorphism was significantly associated with fibrosis severity in women infected with HCV. Regarding ALT and AST levels in HCC group and LC group, there were no significant differences between the studied groups in the level of the two parameters. These results are compatible with Rady et al., (2011).

ACCEPTED MANUSCRIPT These results were incompatible with Kumar et al., (2014) who reported significant differences in ALT and AST levels between chronic hepatitis patients with and without HCC. These findings suggest that serum ALT levels could not predict the histological damage of livers infected with HCV, which was in agreement

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with the results of Liu et al., (2009) who concluded that though a serum ALT level can correlate with the grades of liver necro-inflammatory

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activity, it cannot serve as a parameter to assess the liver damage of

SC

patients with chronic hepatitis C.

In the current study, there was no significant correlation between

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MPO expression and each of AST and ALT levels in LC patients. These

MA

results are in agreement with the published results by Ali et al., (2009). However, a significant correlation between MPO expression and both AST and ALT in LC patients was documented by Bekheet et al.,

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(2009).

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The diagnosis of HCC without pathologic confirmation is achieved by analyzing serum AFP levels combined with imaging techniques, including

ultrasonography,

magnetic

resonance

imaging,

and

AC C

computerized tomography. AFP is the biomarker most widely used to test for HCC, but the sensitivity and specificity of AFP vary widely, and total AFP is not always specific, especially when HCC is in its early stages (Song et al., 2013). In the present study, AFP level was highly significant increased in HCC group compared to chronic HCV group at P < 0.0001, table (2). This is matched with the study done by Eissa et al., (2013) who found that AFP levels were significantly higher in patients with HCC than liver cirrhosis patients.

ACCEPTED MANUSCRIPT In the present study, HCC patients were subdivided according to the level of AFP into above and below 200 ng/ml, this was done according to Leerapun et al., (2007). In the current study that, there is100% of cirrhotic group and 68% of HCC patients group had AFP serum levels less than 200 ng/ml. On the other hand, 32% of HCC patients had AFP serum levels more than 200 ng/ml, table (3). Similar

PT

studies reported that AFP serum levels above 200 ng/mL are highly

RI

specific for HCC diagnosis in patients with cirrhosis and coinciding

SC

radiologic evidence of focal hepatic lesions (Bruix and Sherman, 2005). Daniele, Farinati and their colleagues showed that up to 50%

2004 and Farinati et al., 2006)

NU

of patients with HCC have an AFP level below 20 ng/mL (Daniele et al.,

MA

Johnson (2001) mentioned that elevated levels of AFP can also be found in patients with non-malignant chronic liver disease, including 15-

ED

58% with chronic hepatitis and 11-47% with liver cirrhosis, Thus, AFP cannot be used as a sole tool to screen for and diagnose HCC. New

EP T

reliable serum biomarkers need to be identified soon to complement AFP in order to improve clinical outcomes for patients.

AC C

This study revealed non-significant distribution between MPO expression and tumor size and site of tumor in HCC group. Also, there was none -significant distribution between vascular invasion and grade of tumor in HCC group. Searching in literature proved that no previous studies were done to detect the correlation between MPO expression and different clinicopathological features of HCC patients. Concerning expression of MPO in liver cirrhosis patients, this study revealed that MPO was expressed in various cells in the liver. MPO was identified in kupffercells (KCs),

ACCEPTED MANUSCRIPT polymorph nuclear leukocytes (PNLs), fibrous tissue septa and hepatocytes. In the present study, Immunostaining revealed positive MPO expression in kupffer cells of almost all studied liver biopsies of LC patients (82.4%), table (7). Kupffer cells represent approximately 10% of

PT

the liver cells and 80% of the tissue macrophage population and play an important role in the defense mechanisms of the body (Amanzada et al.,

RI

2011).

SC

As long as the KCs were immunostained positive for MPO, this identifies the involvement of MPO in HCV related liver insult and

NU

inflammatory process. These results agreed with the studies done by Nahon et al., (2009) and Rensen et al., (2009). These results did not

MA

confirmed by a study done by Amanzada et al., (2011), who reported that MPO is not detectable after the differentiation of monocytes into

ED

macrophages.

EP T

Increased MPO expression has a pathogenic role in the progression of liver dysfunction through hepatic endothelial cell damage. The respiratory burst of Kupffer cells adequately explains the oxidative

AC C

damage that is observed in chronic liver disease. Kupffer cells express MPO that may be an important source of oxidative damage during chronic liver injury.

Oxidative free radicals play a role in liver cell

damage and apoptosis through impairment of antioxidant enzymatic defense system (Ali et al., 2009). In the present study, immunostaining revealed few (3%) MPO expression in PNLs in LC patients, table (12). However, Arnhold (2004) reported that azurophilic granules of PNLs contain a huge amount of MPO.

ACCEPTED MANUSCRIPT MPO is involved in the formation of reactive oxygen species and oxidation of biological material together with the membranous NADPH oxidase. In stimulated PNLs, NADPH oxidase reduces molecular oxygen to superoxide anion radical. This species and its dismutation product, hydrogen peroxide, are substrates for MPO (Arnhold, 2004). Nahon and his colleagues also mentioned that Activated macrophages and

PT

neutrophils release MPO into the extracellular space. MPO adheres to cell

RI

membranes and forms hypochlorous acid, which easily crosses the plasma membrane, to deplete intracellular glutathione. The MPO

SC

generated hypochlorous acid also damages DNA bases and impairs

NU

nucleotide excision repair, thus favoring the appearance of somatic mutations and development of a malignant clone (Nahon et al., 2009). this

work,

Immunostaining

MA

In

revealed

myeloperoxidase

expression in fibrous tissue septa LC patients (70.6%), table (7). This

ED

result was in accordance with Lonardo et al., (2004) who stated that hepatic fibrosis was associated with increased number of MPO-

EP T

expressing macrophages in connective tissue septa. This explains the role of free radicals produced by MPO in the pathogenesis of liver cirrhosis. Myeloperoxidase may stimulate production of extracellular matrix

AC C

by kupffer cells. Alteration of extracellular matrix has been linked to the activation of hepatic stellate cells, which is a critical step in the development of hepatic fibrosis.It was found that hepatitis C virus infection is characterized by remarkable levels of oxidative stress (Bekheet et al., 2009). In a study carried out by Ali et al., (2009), the serum myeloperoxidase activity was significantly elevated in chronic and cirrhotic hepatitis compared to the healthy group. Also there was a highly

ACCEPTED MANUSCRIPT significant elevation in MPO activity in the patients with cirrhotic HCV compared to the patients with chronic HCV, indicating its usefulness as both diagnostic and prognostic hepatic biomarker in hepatitis C infection. In

the

current

study,

immunostaining

revealed

(38.2%)

myeloperoxidase expression in hepatocytes of LC patients, table (7). This

PT

was similar to the study done by Bekheet et al., (2009).

RI

Concerning the expression of MPO in HCC and LC patients; the present study revealed more MPO expression in HCC patients when

SC

compared with LC patients, table (18, 20, 22, and 24). This result is similar to those studies done by Do Carmo et al., (2012) and Nahon et

NU

al., (2012). Myeloperoxidase levels and oxidative-stress markers are elevated in malignant tissues, suggesting a role for MPO-derived oxidants

MA

in carcinogenesis. An important MPO product, hypochlorous acid, has been associated with bases changes in genomic DNA, through oxidation

ED

and chlorination mechanisms. The incorporation of these nucleosides into the host DNA may cause transition mutations in genes, resulting in

EP T

alteration of DNA repair, DNA replication and regulation of the cell cycle. HCV disturbs the redox balance of the cells promoting the production of ROS and triggering the anti-oxidant defense such as the

AC C

activation of catalase and glutathione systems (Do Carmo et al., 2012). Do Carmo et al., (2012) found that the levels of MPO in the plasma of patients infected with HCV-HCC were higher in comparison to patients with chronic hepatitis C or with those patients with severe fibrosis indicating that MPO could be a suitable candidate to HCC screening in patients infected by HCV. Nahon et al., (2012) also mentioned that myeloperoxidase was associated with high risk of HCC.

ACCEPTED MANUSCRIPT Galli et al., (2005) and Osterreicher et al., (2005) reported that oxidative stress plays a central role in the progression of hepatitis to cirrhosis. Also, it plays an important role in the pathogenesis of liver failure. Furthermore, oxidant production associated with viral hepatitis leads to mutations in DNA and RNA, which subsequently leads to hepatocellular carcinoma. Schults et al., (2012) demonstrated that

PT

increased expression of MPO in the liver correlated with decreased

RI

nucleotide excision repair (NER) capacity within the liver. Individuals with a reduced NER pathway capacity are at greater risk to develop

SC

cancer as NER is the primary pathway that removes the damage caused

NU

by DNA helix distorting lesions, when DNA damage is not effectively removed, these lesions can be highly mutagenic. Finally; the present

MA

study showed that marked expression of MPO plays an important role in the pathogenesis of HCV-associated HCC and it occurs in an early stage of hepatocarcinogenesis. This study could provide valuable, predictive

AC C

EP T

ED

parameters that can be used clinically in the prognosis of HCC patients.

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Detection of BCL2 polymorphism in patient with hepatocellular carcinoma. Am J Cancer Prevention. 2015; 3(2):27-34.

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[3] Abdel-Wahab M, El-Ghawalby N, Mostafa M, Sultan A, Ezzat F, Mansoura

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et al. Epidemiology of hepatocellular carcinoma in Lower Egypt, Center.

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2007; 54(73): 157-162.

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antioxidant of arylesterase and paraoxonase as biomarkers in patients with hepatitis C virus. Clinical biochemistry. 2009 Sep

ED

1;42(13-14):1394-400.

[5] Amanzada A, Malik IA, Nischwitz M, Sultan S, Naz N, Ramadori

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G. Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflammed liver. Histochem Cell Biol. 2011 Mar 1;135(3):305-315.

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[6] Arnhold J. Properties, functions, and secretion of human myeloperoxidase. Biochemistry (Moscow). 2004 Jan 1;69(1):4-9.

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Kumar S, Chawla YK, Ghosh S and Chakraborti A. Severity

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1;50(5):1484-93. Nahon P, Zucman-Rossi J. Single nucleotide polymorphisms

hepatology. 2012 Sep 1;57(3):663-74.

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Moreau R, Ganne-Carrié N, Grando-Lemaire V, N’Kontchou G, Trinchet JC, Pessayre D. A variant in myeloperoxidase promoter HCV-related

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1;56(2):426-32.

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Rady MA, Sabawi M, Tharwa E, Hassouna M, Thabet YF,

et al. Significance of double murine minute protein and P53 gene polymorphism in development of hepatitis C virus-related hepatocellular carcinoma. MedicinaInterna. 2011; 8: 1-12.

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Song P, Gao J, Inagaki Y, Kokudo N, HasegawaK, et al.

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Biomarkers: Evaluation of Screening for and Early Diagnosis of Hepatocellular Carcinoma in Japan and China. Liver Cancer. 2013; 2: 31-39.

ACCEPTED MANUSCRIPT Table 1 Age and Gender distribution among studied patients.

Cirrhosis (n=34)

(47-75) 60.12 ± 1.799

(42-70) 55.79 ± 1.344

19(76%) 6 (24%)

28 (82.35%) 6 (17.65%)

PT

Age (years) Range Mean ±SEM Gender Male: (%) Female: (%)

HCC (n=25)

94-410 170 178.5± 16.58

< 0.0001

NU

1-10 4 4.779 0.5534

SC

P-value

MA

±

17 (68%) 34 (100%) < 0.0001 134.4 ± 4.779 ± 8.976 0.5534 8 (32%) 272.3± 26.59

0 (0%) 0

< 0.0001

EP T

AFP < 200 ng/ml N (%) Mean ±SEM AFP > 200 ng/ml N (%) Mean ±SEM

HCC n=25 LC n=34

ED

AFP (ng/ml) Range Median Mean ±SEM

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Table 2 AFP serum levels in the studied groups.

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Table 3 Mean values of AST and ALT among studied cases.

Parameter

HCC (N=25)

LC (N=34)

P-

Mean ±SEM

Mean ±SEM

AST

61.88 ± 5.790

57.32 ± 4.271

0.5195

ALT

64.20 ± 6.495

60.71 ± 5.053

0.6681

value

P > 0.05: Non significant (NS), P ≤ 0.05: Significant (S).

ACCEPTED MANUSCRIPT Table 4 Myeloperoxidase tissue expression in various locations in cirrhotic cases.

Liver cirrhotic Cell

MPO tissue expression

-ve expression No (%) Kupffer cell 6 (17.6) %

+ve expression No (%) 28(82.4) %

PNL in 31(91.2)% sinusoids Fibrous 10(29.4)% tissue septa

3(8.8)%

Hepatocyts

13(38.2)%

P value < 0.0001

RI

21(61.8)%

PT

24(70.6)%

significant

SC

χ 2 = 44.94

NU

Table 5 Relation between Myeloperoxidase expression and the gender of the HCC patients.

MPO tissue expression

MA

Clinic-pathological features No of patients -ve

AC C

Male Female

χ 2 = 0.1628

No. (%)

No. (%)

25

3(12)%

22(88)%

19(76)%

2(8)%

17(68)%

6(24)%

1(4)%

5(20)%

ED

Gender

EP T

Total number

+ve

P- value = 0.6866

ACCEPTED MANUSCRIPT Table 6 Relation between Myeloperoxidase expression and the gender of cirrhotic subjects.

MPO expression

patients -ve

+ve

No. (%) 34

6

Male

%) 28

Female

(82 6 (18 %) 22 (64 %)

%)

0 (0%)

AC C

EP T

ED

χ2= 1.561

6 (18%)

P- value=0.2115

MA

6 (18%)

RI

number

(17.6 28 (82.4 %)

NU

Total

No. (%)

PT

of

SC

Gender

No.

tissue

ACCEPTED MANUSCRIPT Table 7 Relationship between Myeloperoxidase tissue expression and serum AFP level in HCC Patients.

Serum AFP

MPO tissue expression -ve

+ve

No. (%)

No. (%)

Total

14 (56%)

17 (68 %)

>200 ng/ml 0 (0 %)

8 (32%)

8 (32 %)

`Total

22 (88 %)

25 (100 %)

RI

P- value=0.2053

AC C

EP T

ED

MA

NU

SC

χ 2 = 1.604

3 (12 %)

PT

<200 ng/ml 3 (12 %)

ACCEPTED MANUSCRIPT Table 8 Relation between Myeloperoxidase expression and biochemical parameters in HCC subjects.

Parameter

HCC subjects

P- value

-ve

Moderate

Marked

No.= 3

No.= 7

No.= 15

Range

(30-44)

34-108

28-150

Median

36

60

57

RI

ALT (U/L) 35-36

40-180

Median

36

60

NU

Range

30-110 66

MA

± 35.67± 0.333 80.57± 18.72 62.27 ± 5.439

AC C

EP T

ED

Mean SEM

0.2496

± 36.67± 4.055 60.71± 9.588 67.47± 8.098

SC

Mean SEM

PT

AST (U/L)

0.1240

ACCEPTED MANUSCRIPT Table 3 Relation between Myeloperoxidase expression and biochemical parameters in cirrhotic subjects.

Parameter

Liver cirrhosis subjects (kupffer cell) Mild

Moderate

Marked

No.=12

No.=13

No.=3

Range

(30-150)

28-81

44-64

Median

71.5

44

50

Pvalu e

RI

± 68.75 ± 9.061 46.69 ± 4.374 52.67± 5.925

AC C

EP T

ED

MA

NU

SC

Mean SEM

PT

AST (U/L) 0.08 29

ACCEPTED MANUSCRIPT Table 4 Degree of expression of Myeloperoxidase in kupffer cell of cirrhotic subjects compared to HCC subjects.

MPO tissue expression Moderate( Marked(++ ++) +)

LC(k 6 (10 12 (20.5 13 (22 %) upffe %) %) r) 3 (5 %) 0 (0 %)

7 (12 %)

3 (5 %)

NU

Total 9 (15 12 (20 %) 20 (34 %) %) χ 2= 21.94

AC C

EP T

ED

MA

P- value< 0.0001

34 (57.5 %)

15 (25.5 %) 25 (42.5%)

SC

HCC

No. (%)

PT

No. (%)

RI

Negativ Mild (+) e No. (%) No. (%)

Total

18 (31 %)

59 (100 %)

ACCEPTED MANUSCRIPT Table 5 Comparison between degree of expression of Myeloperoxidase in (PNL) in sinusoids of cirrhotic and HCC subjects.

MPO tissue expression

Total

Negative

Mild (+)

Moderate(++) Marked(+++)

No (%)

No (%)

No (%)

No (%)

31 (52.5)% 1(2%)

2(3)%

0(0)%

HCC

3(5)%

0(0)%

7(12)%

Total

34(57.5)% 1(2)%

9(15)%

LC

EP T

ED

MA

NU

P< 0.0001

AC C

RI

15(25.5)%

SC

χ 2 = 41.43

PT

(PNL)

15(25.5)%

34(57.5)%

25(42.5)% 59(100)%

ACCEPTED MANUSCRIPT Table 6 Degree of expression of Myeloperoxidase in fibrous tissue septa of LC subjects compared to HCC subjects.

MPO tissue expression

9 (15 %)

7 (12 %)

HCC

3 (5%)

0 (0 7 (12 %) %)

Total

13(22%) 9 (15 %)

χ 2= 13.85

P= 0.0031

8 (13.5 %)

MA

NU

14 (24 %)

ED EP T AC C

PT

LC in 10 fibrous (17%) tissue septa

34(57.5%)

15 (25.5 %)

25(42.5%)

RI

No (%)

Mild Moderate(++) Marked(+++) (+) No (%) No (%) No (%)

SC

-ve

Total

23 (39 %)

59(100 %)

ACCEPTED MANUSCRIPT Table 7 Degree of expression of Myeloperoxidase in hepatocytes of cirrhotic subjects compared to HCC subjects.

MPO tissue expression

LC in 21(35.5%) 6 hepatocyts (10 %)

5 (8.5 %)

3 (5%)

0 (0 7 (12 %) %)

Total

13(22%)

9 (15 %)

NU MA

P <0.0001

14 (24 %)

AC C

EP T

ED

χ 2= 29.08

2 (3.5 %)

34(57.5%)

15 (25.5 %)

25(42.5%)

SC

HCC

PT

No (%)

Mild Moderate(++) Marked(+++) (+) No (%) No (%) No (%)

RI

-ve

Total

23 (39 %)

59(100 %)

ACCEPTED MANUSCRIPT

AC C

EP T

ED

MA

NU

SC

RI

PT

Table 1 Age and Gender distribution among studied patients. ............................................................. 36 Table 2 AFP serum levels in the studied groups. ................................................................................. 36 Table 3 Serum AFP level in HCC cases....................................................... Error! Bookmark not defined. Table 4 Mean values of AST and ALT among studied cases. ................................................................. 36 Table 5 Myeloperoxidase tissue expression in various locations in cirrhotic cases................................. 37 Table 6 Relation between Myeloperoxidase expression and the gender of the HCC patients. ................ 37 Table 7 Relation between Myeloperoxidase expression and the gender of cirrhotic subjects. ............... 38 Table 8 Relationship between Myeloperoxidase tissue expression and serum AFP level in HCC Patients..................................................................................................................................... 39 Table 9 Relation between Myeloperoxidase expression and biochemical parameters in HCC subjects. .......................................................................................................................................... 40 Table 10 Relation between Myeloperoxidase expression and biochemical parameters in cirrhotic subjects. ............................................................................................................................. 41 Table 11 Degree of expression of Myeloperoxidase in kupffer cell of cirrhotic subjects compared to HCC subjects................................................................................................................. 42 Table 12 Comparison between degree of expression of Myeloperoxidase in (PNL) in sinusoids of cirrhotic and HCC subjects. ............................................................................................................ 43 Table 13 Degree of expression of Myeloperoxidase in fibrous tissue septa of LC subjects compared to HCC subjects................................................................................................................. 44 Table 14 Degree of expression of Myeloperoxidase in hepatocytes of cirrhotic subjects compared to HCC subjects................................................................................................................. 45

ACCEPTED MANUSCRIPT Highlights:

 Research in genomics represents the present and future in Hepatocellular carcinoma. 

Emerging clinical and preclinical evidence suggesting involvement of myeloperoxidase in Hepatocellular carcinoma and clinical response.

markers may help us identify high risk patients.

PT

 Immunohistochemical expression still work in progress, but these new

AC C

EP T

ED

MA

NU

SC

improvement of HCV associated HCC .

RI

 Role of immunohistochemical expression of myeloperoxidase in the