Expression of hnRNPK & Claudin-4 in HCV-Induced Early HCC and Adjacent Liver Tissue

BACKGROUND: HCC in Egypt usually occurs in HCV cirrhotic livers with poor prognosis due to late diagnosis. High hnRNPK & low Claudin-4 profiles indicate Epithelial Mesenchymal Transition (EMT), malignant transformation and high-grade tumours. AIM: We studied the immunohistochemical expression of hnRNPK and Claudin-4 in HCV induced early HCC (eHCC) and adjacent liver tissue in Egyptian patients to improve eHCC detection in cirrhotic livers with better curative therapy options. METHOD: We studied the immunohistochemical expression of hnRNPK and Claudin-4 in 100 Egyptian patients resection specimens of HCV induced early HCC (eHCC) and adjacent liver tissue, in order to improve eHCC detection in cirrhotic livers, thus improving their therapeutic options. RESULTS: Early HCC grade significantly directly correlated with nuclear hnRNPK/5HPFs count and inversely correlated with Claudin-4 expression %, with a converse correlation between hnRNPK and Claudin-4. Moreover in eHCC, combined hnRNPK ≥ 30/5HPFs & Claudin-4 ≥ 40% significantly distinguished low grade eHCC (G1) from high grade eHCC (G2&G3), with sensitivity 97% & specificity 69.7% for hnRNPK ≥ 30/5HPFs, and with sensitivity 70% & specificity 94.3% for Claudin-4 ≥ 40%. Moreover in the adjacent liver, both markers expressions significantly directly correlated with each other and with METAVIR fibrosis score but not with activity. Furthermore, 58% of eHCCs showed hnRNPK ≥ 30 Claudin-4 < 40% profile, indicating EMT type 3, compared to 26% with hnRNPK ≥ 30 Claudin-4 ≤ 10% profile in adjacent cirrhotic/ precirrhotic liver, with significant use of combined hnRNPK ≥ 30/5HPFs & Claudin 4 ≤ 10% as eHCC prediction cut offs in cirrhosis (p < 0.05). CONCLUSION: Combination of hnRNPK and Claudin-4 can indicate early HCC development in HCV cirrhotic livers using hnRNPK ≥ 30/5HPFs & Claudin-4 ≤ 10% cut offs. Also, combination of hnRNPK ≥ 30/5HPFs & Claudin-4 ≥ 40% can distinguish low grade eHCC (G1) from high grade eHCC (G2&G3).


Introduction
In E gypt, HCC is one of the commonest cancers [1][2]. It occurs with cirrhosis [3] since it leads to alteration of hepatocyte proliferation and promotion of tumorigenesis [4][5][6]. Early HCC treatment is curative [3]. Nevertheless, usually, HCCs have a poor prognosis due to late diagnosis and lack of effective therapy options [6]. Accordingly, early detection of HCC in cirrhotic patients is mandatory [3].
Heterogeneous nuclear RNA-binding proteins (hnRNPs) are crucial for RNAs cont rol, mRNA export, turnover, localization, and translation [7][8]. Their aberrant ex pression is associated with cancer c ell proliferation, angiogenesis, invasion, epithelial mesenchymal transition (EMT) and metastasis [8]. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a potential tissue biomarker for early detection of HCC [8][9]. It is a DNA & RNA binding protein [10] and contributes to chromatin remodeling, transcription and translation [11]. It is distinguishable from the other hnRNPs by its capability to interact with numerous proteins through its K interactive region [12][13] that fits it in the center of a network to influence diverse cellular processes. HNRNPK is a potential tumor suppressor [10]. Its knockout results in reduced survival and increased tumorigenicity [12][13]. Its overexpression is associated with poor clinical status [10]. http://www.mjms.mk/ http://www.id-press.eu/mjms/ In hepatitis, hnRNPK is import ant for HCV pathogenesis [3,8,9]. It shows similar sequences with HCV core protein binding domain [3]. Moreover, hnRNPK is involved in the multistep process of hepatocarcinogenesis of bot h HBV replication and HCV pathogenesis with eventual cirrhosis and HCC [3], [8]. In this context, an increase of HCC in Egypt is due to high HCV prevalence particularly in cirrhotic patients compared to the declining incidence of HBV, since Egypt exhibits the highest HCV prevalence worldwide [14][15][16].
In HCC, hnRNPK overexpression is a marker for HCC [ 3,11]. Positive tissue hnRNPK staining is an indicator of HCC and facilitates accurate early HCC distinction from high-grade dysplasia and other small nodules, which c an be extremely challenging [3]. Moreover, hnRNPK expression in the early and late HCC is reported to be > 3 folds higher and stronger than adjac ent cirrhotic [3] or normal liver [16], due to the nuclear shift of K protein from the cytoplasm into the nucleus in tumours.
In addition, hnRNPK overexpression significantly correlates wit h the increased t umour size, active tumour growth, intrahepatic micrometastasis and microsatellite nodules formation [3].
In the same context, increased nuclear levels of K protein in c ancers plays a role in the altered telomeric processes [17], exerts antiapoptotic effect on cancer cells independently of p53 status [11], activates c-myc promot er in hepatocytes in response to mitogens and following liver injury [17], and activates VEGF transcription by selective binding to VEGF promoter [8]. Since Epithelial Mesenchymal Transition (EMT)-angiogenesis-stem cell-like crosstalk is a key factor for HCC [18], the t rans formed tumour cells acquire stem cell features, show multidrug resistance, and induce local recurrence [18][19], metastasis & cancer progression [ 20]. Also, hnRNPK silencing significantly decreases EMT phenotype in cancer cells [8,21].
In this regard, loss of critical junction proteins -including Claudins -leads to loss of epit helial c ell adhesion, thus repres ents the first step of EMT [22]. Claudins are trans membrane proteins and important components of tight junctions (TJs) [23][24] that act as cell adhesion molecules, thus preserving cohesion in the tumour mass, suppressing c ell proliferation & tumorigenesis, and function as cell migration barrier [24]. Moreover, Claudine expression patterns affect the tumour behavior [23]. Downregulation of s everal Claudins in cancer is consistent with the disruption of TJs during tumourigenesis [23] since low expression or loss of TJs is associated with malignant transformation and characterizes the highly metastatic cancers [23].
Since markers combination improves diagnosis [3], we studied the immunohistochemic al expression of hnRNPK and Claudin-4 in HCV induced early HCC (eHCC) and adjacent liver tissue in Egyptian patients to improve eHCC detection in cirrhotic livers with better curative therapy options.

Material and Methods
The study was held on 100 HCC resection specimens with a history of HCV infection, obtained retros pectively from archival paraffin blocks at pathology department Theodeor Bilharz Res earch Institute (TB RI) (2010-2015).
A-Inflammatory activity and fibrosis in the adjacent liver tissue were evaluated using ME TAVIR scoring system [32] as follows:

C-Immunohistochemical technique:
Immunohistochemistry for hnRNPK & Claudin-4 was performed on tumours and adjacent non tumorous tissue sections cut from the paraffin blocks and stained with anti-human hnRNPK & Claudin-4 monoclonal primary antibodies (Santa Cruz Biotechnology, CA, USA) at 1:150 dilution. Slides were sectioned at 4μm onto positively charged slides (Superfrost plus, Menzel-Glaser, Germany) and the slides were stained on an automated platform the (Dak o Autostainer Link 48). Heat induced antigen retrieval was used for 30 min at 97°C in the high-PH EnVision™ FLE X Target Retrieval Solution, and the primary antibody was used at a dilution of 1 in 100. The det ailed histopathological assessment was done regarding confirmation of diagnosis and grading of malignant cases.

D-
E valuation of the immunostaining:  HNRNPK: Only nuclear staining was counted per 5 High Power Fields (5HP Fs). Positivity cut off at 10% was established.
 Claudin-4: Semiquantitative H score for staining intensity and % of expression was used. Positivity cut off at >10% was established [35].

Stati stical analysi s
SPSS software version 18 was used for data management and analysis. Quantitative data were presented as mean ± SD. Qualitative data were presented as frequencies and percentages. Spearman's correlation coefficient was calculated to assess the relationship bet ween variables. Tests were considered statistically significant when P< 0.05. Cut off values for both markers were chos en with sensitivity and specificity evaluation.

B-hnRNPK:
Cytoplasmic and nuclear hnRNPK expression in eHCC & adjacent liver was noticed. Only the nuclear expression was counted (Fig. 2). Our study showed that all of the non tumorous liver tissue adjacent t o HCC significantly exhibited increased nuclear hnRNPK from a mean nuclear expression value 23.86 ± 7.587/5HPFs to 40.14 ± 17.05/ 5HPFs in HCC (P < 0.001) (Tables 1 & 2; Fig.  3a).

III -Regarding METAVIR fibrosi s & activity scores in adjacent non-tumorous liver ti ssue:
Most of our cases showed pre-cirrhotic (F3) rather than complete cirrhotic nodules (F4) due to the clinical difficulty of obtaining liver biopsies in cirrhotic patients.
Nevertheless, neither hnRNPK nor Claudin-4 showed a significant difference or correlation with inflammatory activity scores (Tables 1-3). Furthermore, both markers neither correlated with age or gender (Table 3).
In this study, both cytoplasmic and nuclear hnRNPK expressions in HCC as well as in the adjacent liver were noticed. Despite that cytoplasmic hnRNPK indicat es its overexpression [10], nuclear hnRNPK was also reported to be higher in proliferating compared to resting hepat ocytes [3,17], which was similar to our findings. Moreover, nuclear http://www.mjms.mk/ http://www.id-press.eu/mjms/ hnRNPK level was reported to be higher in neoplasms than in adjacent normal parenchyma in contrast to the cytoplasmic hnRNPK that remains unchanged in both neoplastic and surrounding tissues [17]. Therefore we counted only nuclear hnRNPK in our study. Furthermore, since several positivity cut offs were identified for early and lat e HCCs [3], and to avoid tissue variations, we used hnRNPK nuclear count > 10/5HPFs as positivity cut off.
In addition, stronger nuclear hnRNPK was reported in HCC in comparison to faint er nuclear staining in cirrhosis [3]. This is due to hnRNPK translocation into the nucleus [3,17], which reflects its involvement in altered DNA and/or RNA in malignancy [17]. Nevertheless, our study showed rather moderate to strong nuclear hnRNPK in adjacent cirrhotic / precirrhotic liver, with a significant increase of the mean nuclear hnRNPK count in HCCs compared to adjacent cirrhotic liver, confirming the c ritical role of hnRNPK in hepatocytes proliferation, differentiation and tumorigenesis promotion in cirrhotic liver [10].
Regarding Claudin-4, all of our caseswhet her HCC or adjacent liver-showed low Claudin-4 expression profile (weak & negative cytoplasmic staining compared to the moderat e and strong expression in bile ducts) since bile ducts used as an internal control as mentioned in Holczbauer et al., 2013 [27] study. In the same context, absent Claudin-4 expression in non tumorous hepatocytes & HCC was reported, in contrast to normal cholangiocytes and cholangiocarcinomas [27]. None of our cases exhibited high Claudin-4 expression profile. Coming along with Konstantinos et al., 2014 [35], this indicates molecular down regulation and subs equent high recurrence and low diseas e free survival rates, hence pointing to type 3 EMT [18,36].
Nevertheless, our study significantly showed increased Claudin-4 expression in HCC compared to the adjac ent liver, in whic h weak cytoplasmic staining was detected. This came similar to Konstantinos et al., 2014 [35] where up regulation of Claudin-4 and other proteins in HCC was mentioned. In the same context, Holczbauer et al., 2013 [27] showed similar findings in some of their cases where Claudin-4 expressed by the apical poles of the glandular and alveolar forms of HCC. Also, Konstantinos et al., 2014 [35] reported -as other Claudins-, cytoplasmic staining pattern represented a loss of function & intracellular localization of Claudins, thus pointing to type 3 EMT [18,36].
Despite that 50-60 years is the most frequent age range for HCC in Egypt [38], and despite association of inc reased Claudin-4 expression with female gender [37], our study showed no significant correlation between age, gender and both markers expression.
Regarding the adjacent liver, the majority of our cases showed F3 fibrosis. In this context, it was reported that developing HCC without advanced fibrosis (F4) may be due to other factors in the pathogenesis of HCV [38]. In E gypt, despite the high incidence of HCC in cirrhosis, HBV infection (whether occult or combined HCV HBV infection forms ), diabetes and smoking have synergistic effects in HCC development [14]. Nevert heless, most of our cases showed pre-cirrhotic (F3) rather than cirrhotic F4 due to the clinical difficulty of obtaining liver biopsies from cirrhotic patients.
In our study, the mean expression of both markers significantly directly correlated wit h each other and with ME TAV IR fibrosis score but not inflammatory activity, with significant use of bot h of hnRNPK > 30/5HPFs & Claudin-4 < 10% cut offs (P < 0.05). The majority expressed Claudin -4 < 10% particularly at the periphery of cirrhotic nodules. Similarly, according to Holczbaueret al., 2014 [27], it is due to bile duct proliferation [27], and according to Tsujiwaki et al., 2015 [28] is due to increased proliferation of progenit or cells that express Claudin-4, thus suggesting subsequent differentiation into mature hepatocytes.
Nevertheless, majority of adjacent cirrhotic / precirrhotic liver (68% ) expressed hnRNPK < 30/5HPFs Claudin-4 < 10% profile in contrast to 26% for hnRNPK > 30/HPFs Claudin-4 < 10% profile suggesting type 2 EMT as healing and regenerative process [18]. This also indicates that not all EMT in cirrhosis undergo malignant transformation into HCC particularly that the majority of those cases showed F3 pre-cirrhotic rather than F4 complete cirrhotic changes. Therefore it becomes compatible with incomplet e rather than complete EMT with a chance for reversal and healing [36] instead of malignant progression. This helps in identification of the leading factors of progression through finding of targeted therapeutic approaches to suppress nuclear hnRNPs translation [8,11].