P- element Induced Wimpy Testis (PIWI), a Potential Candidate Marker for Hepatocellular Carcinoma

doi:10.21203/rs.3.rs-1889286/v2

P-Element-induced wimpy testis (PIWI) proteins are engaged in epigenetic regulation of gene expression in germline cells when they are in combination with PIWI-interacting RNA (piRNA). Different types of tumour cells have been found to exhibit abnormal expression of piRNA and PIWI proteins. HCC is majorly associated with lower survival rates. Thus, there is an urgent need for improve less invasive diagnostic methods. Purpose: The aim of this study was to assess the diagnostic potential by determining the expression profiles of PIWI-like protein-1, -2, -3, & -4 (PIWIL1, PIWIL2, PIWIL3, & PIWIL4) in hepatocellular carcinoma, and determine its association with clinicopathological features. Methods: Samples from patients comprising serum and frozen fragments of paired tissue specimens (tumour and adjacent non-malignant liver tissue) were employed for molecular analyses. Real-time polymerase chain reaction was performed on 50 tissue and serum samples from HCC patients and 25 serum samples from healthy controls. Results: Expression levels were significantly higher in both HCC tissue and serum samples than in the controls (p<0.001). Additionally, the diagnostic performance was assessed by ROC curves, the PIWIL1-4 levels in serum showed better potential and sensitivity to screen HCC patients than their levels in tissues. Moreover, some PIWIL elements were found here to be significantly correlated with the HCC profile. Conclusion: PIWI expression could be considered to be associated with HCC progression, and could be a tool for the diagnosis and prognosis of HCC, additionally, it could represent a possible therapeutic target.

Hepatocellular Carcinoma (HCC)

PIWIL1

PIWIL2

PIWIL3

PIWIL4

qPCR

Molecular non-invasive diagnosis

Liver cancer continues to be a global health problem, with global rates increasing ^{1, 2}. By 2025, the disease is predicted to affect over 1 million people ³. Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer, accounting for around 90% of cases. In Egypt, HCC is seen as a problematic health problem, with the number of patients more than doubling over the last decade ⁴. The most important risk factors for the development and progression of HCC include viral infection Hepatitis B virus (HBV) and hepatitis C virus (HCV). Patients with viral clearance for HCV and HBV who have cirrhosis are thought to be at a high risk of developing HCC ³. Along with mutational changes ascribed to tobacco and aristolochic acid (AA) as probable pathogenetic cofactors in HCC, non-alcoholic steatohepatitis (NASH) is a growing aetiology of HCC ⁵.

HCC is typically diagnosed using non-invasive criteria. Screening and sensitive imaging techniques such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) have advanced dramatically in recent years. These diagnostic tools demonstrate increased detection of HCC at an earlier stage, hence improving patient outcomes ⁶. There is a widely accepted protocol for diagnosing chronic liver disorders (CLD), which involves evaluating liver function using a series of serum-level enzyme assays and a significant tumour marker, α-fetoprotein (AFP). Serum biomarkers have long been used to screen for and diagnose HCC, and AFP is one of the most thoroughly studied biomarkers in solid tumours, inflammatory diseases, and premalignant lesions ⁷. As a result, AFP is the most often used tumour marker for HCC in clinical practise. The sensitivity and specificity of AFP in the early phases of hepatocarcinogenesis vary according to numerous investigations. However, AFP has exhibited suboptimal results in terms of therapeutic surveillance and early identification ⁸. As a result, much effort has been made to develop a reliable molecular marker for HCC in its early stages ⁹. There is a need for more accurate serum biomarkers with increased sensitivity and specificity that complement AFP and improve clinical outcomes for patients.

Recent studies have shown that other proteins could be used as markers for their diagnostic and prognostic values, including P-element-induced wimpy testis (PIWI) proteins ^{10, 11}. Some of these biomarkers have been proposed as potential, accurate, simple and non-invasive biomarkers for early detection of HCC. In humans, PIWI protein family consists of four proteins: PIWIL1/ HIWI, PIWIL2/ HILI, PIWIL3, and PIWIL4/HIWI2 ¹². PIWI family is a class of genes protected during evolution that play a key role in stem cell regeneration, division, gametogenesis, germ cell proliferation and RNA silencing ^{13, 14}. The expression of PIWI proteins has been linked to cancer with characteristics that include maintaining proliferative signalling, evading growth suppressors, activating invasion and metastasis, mediating genomic instability and mutation, and boosting cell growth, to mention a few ¹⁵. The abnormal expression of PIWIs in cancer were first discovered in 2011¹⁶ and the molecular mechanisms underlying PIWI's oncogenic actions are still being studied. Moreover, PIWIs have been found to regulate the appearance of cancer hallmarks ⁵.

Studies have also shown that PIWI proteins are involved in proliferation, apoptosis, metastasis, and invasion of cancer cells and act as potential diagnostic factors and biomarkers of cancer prognosis ¹⁷. Overexpression of PIWIL1/HIWI gene is seen in various cancers, including seminoma cell hyperplasia ¹⁷, oesophageal squamous cell carcinoma, gastric cancer ¹⁸ and pancreatic adenocarcinoma ¹⁹. This study aimed to determine the efficacy of the 4 PIWI mRNA in humans (PIWIL1/HIWI, PIWIL2/ HILI, PIWIL3 and PIWIL4/HIWI2) as diagnostic biomarkers for HCC patients in both tissue and serum, and to correlate their expression with clinicopathological parameters.

Patient and Sampling

The present study was conducted at Theodor Bilharz Research Institute (TBRI), Egypt. Patients who were diagnosed with HCC by multi-slice triphasic CT and increased alpha feto protein levels were selected. Institutional Approval was acquired from the Research Institute office for IRB (NHTMRI-IRB) (Serial:2-2019), and Theodor Bilharz Research Institute (TBRI-IRB). The research was conducted according to the declaration of Helsinki for human subject research guidelines (2013). Prior to enrolment, all patients and controls signed an informed consent form. Participants' personal, clinical, and laboratory data were collected in strict confidence. Samples were collected from 50 HCC patients, and 25 healthy controls, the study was conducted between January and December 2019. For tissue samples 50 patients undergoing liver resection were sampled for tumour and tumour-adjacent samples, the surgery for liver resection was conducted within the department of surgery NHTMRI Hospital, Egypt. Individuals suffering from other liver diseases (e.g., Auto immune hepatitis, Hemochromatosis, Schistosoma), or diseases such as HIV, and ischemic heart diseases were excluded. Patients with HCV who were taking immunomodulatory interferon therapy were also excluded from the research.

Sample collection

Blood was drawn from 50 patients and 25 control individuals, then it was allowed to clot. samples were then centrifuged at 500 xg for 10 minutes. Serum was then centrifuged, aliquoted, and stored at -80°C. Liver tissues were collected from 50 patients (tumorous and non-tumorous) who underwent liver resection to remove the tumour (non-tumorous sections of the liver from those patients served as controls). Liver sections were stored in lysis buffer at -80°C until use.

Biochemical Parameters

Laboratory tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), Bilirubin, albumin (ALB) and alpha-fetoprotein (AFP) were performed for all subjects as routine tests upon admission, AFP was also measured by ELISA using a commercially available kit (ABCAM, AB79801, Cambridge, UK)

RNA extraction

The miRNeasy extraction kit was used to extract total RNA (Qiagen, Valencia, CA). The extraction procedure was conducted according to the manufacturer’s instructions for both tissue and serum samples. Samples were extracted in duplicates, then the quality and concentration of the samples were measured using a NanoDrop-1000c spectrophotometer (Thermo Fisher Scientific, Cinisello Balsamo, Italy).

Quantitative Real-Time Reverse-Transcription (QPCR)

For quantification of PIWI transcripts, the four primer assays were readymade and acquired from Qiagen. The primers included the four isoforms PIWIL1/HIWI; PIWIL2/HILI; PIWIL3, PIWIL4 and all assays were performed according to the manufacturer’s instructors. The real time PCR amplification was performed by QuantiTect SYBR Green PCR Kits (Qiagen, Valencia, CA). 5μl of the samples were used for the QPCR amplification step. All reactions were run in duplicates. Finally, the ΔΔCT method was used for the relative quantification of mRNAs in all samples ²⁰.

Statistical analysis:

To determine the appropriate number of patients, we regressed their values for the patients against control. Prior data indicated that the standard deviation of control is 0.8 and the standard deviation for the regression errors will be 1.9. Regression was at 1.1, 50 study subjects and 25 normal controls will be an appropriate representation to reject the null hypothesis with a probability (power) of 85%. The Type I error probability associated with this test of this null hypothesis is 0.05. A sample size calculation was conducted, and adapted from other research projects involving HCC diagnosis ²¹. The continuous variables were described as mean ± standard deviation (SD) or median and interquartile range (IQR) according to their distribution, which was determined using a normality test. The frequencies and percentage were used for categorical variables. A p value of < 0.05 was considered statistically significant. To compare the means Mann-Whitney U was used. Chi²test or Fisher’s exact test was used to determine the distribution of categorical variables between groups. The diagnostic performance of the studied markers was assessed by receiver operating characteristic (ROC) curves. Logistic regression was conducted to determine the risk level, for HCC patients. Pearson Correlation was done to correlate between the studied parameters with the studied biomarkers.

Patient characteristics

A total of 50 HCC participants were recruited in this study, the patients included 28 males (56%) and 22 females (44%), with a mean age of 57.2±8.1 and 25 healthy individuals with no history of liver disease or alcohol consumption were included as controls. Individual demographic and clinical data of the studied groups are shown in (Table 1). The results for biochemical parameters were represented as mean±SD for these tests, and had values of 61.4±15.5, 65.8±16.6, 2.3±1.0, 2.9±1.1, 0.9±0.4 respectively, AFP however was 75.0 (40.0- 150.0).

By computed tomography, the number of tumour masses was detected with mean of 1.1±0.2, while tumour size and steatosis were found at 2.25 (0.75- 4.25) and 0.02 (0.02- 0.04) respectively. Regarding pathological diagnosis, 29(38.7%) were diagnosed GI, 11 (14.7%) were with GII and 10 (13.3%) were GIII, 30 (60.0%) were with acinar pattern tumour, 17 (34.0%) with solid tumour and only 3 patients (6.0%) were with acinar/solid tumour. For tumour staging 8 patients (16%) were fibrotic with the predominant number for the cirrhotic patients 42 (84%). Histopathological examination was performed using METAVIR scoring system to determine the hepatitis activity index (HAI), 15 (30.0%) patients were rated as AI, 35 (70.0%) were A2 while none of the patients were A3. Abdominal ultrasounds detected hepatomegaly in 9 (18%) patients, ascites in 20 (40%) patients and splenomegaly in 13 (26%) patients and finally 29 (58.0%) out of the 50 HCC patients were detected with oedema lower limbs (Table 1).

Biochemical Assessments

The biochemical parameters ALT, AST, Bilirubin, ALB and AFP were performed for all subjects as routine tests, their results can be found in the supplementary table (Table 13). Correlation between these results and the expression of PIWIL mRNA, Pearson Correlation was conducted, and no significant difference was found (Supplementary Data: Table 13).

Expression of PIWIL’s in Serum & Tissue.

To assess the significance of expression for PIWIL1, PIWIL2, PIWIL3 and PIWIL4 RNA in the serum of HCC patients, results showed high significance between their levels in patients and healthy controls (p<0.001) (Figure 1 a, b, c and d). In addition to patients’ tissue expression (p<0.01) (Figure 2 a, b, c and d) between tumorous and adjacent nontumorous regions. This indicates that there was an overexpression for all assessed PIWIL mRNAs in both the serum and tissue samples of patients.

Logistic regression analysis of PIWIL’s

To assess the relative risk of HCC presented from PIWIL mRNA, logistic regression analysis model was performed (Table 2). It revealed that PIWIL1-4 is significantly associated with increased risk for HCC in serum (p<0.001) of HCC patients, and in tissue samples of patients (p<0.01).

Diagnostic performance:

Receiver Operating characteristic (ROC) analysis is essential for evaluating the performance of diagnostic tests, and the accuracy of a statistical model. In our investigation, ROC Curve was conducted to assess the diagnostic performance of PIWIL1, PIWIL2, PIWIL3 and PIWIL4 in both serum, and tissue samples of HCC patients, to evaluate their specificity and sensitivity in HCC prediction and to evaluate their discriminatory properties for the patients and healthy individuals. Analysis of the diagnostic value of the 4 PIWI-RNAs showed promising results, indicating that they are associated with convenient disease characteristics and could differentiate between serum of HCC patients with those of controls. For discrimination, it was found that the 4 PIWIL mRNAs were with sensitivity of 100% and specificity of 100% with an area under curve (AUC) of 1.0 (p <0.001, 95% C.I: 1.0 – 1.0).

Regarding the diagnostic performance of the 4 PIWILs RNA in HCC tissue, the analysis revealed statistical significance with PIWIL1, it was with sensitivity of 80.0% and specificity of 72.0% with an area under curve (AUC) of 0.80 (P < 0.001, 95% C.I: 0.71 – 0.89). For PIWIL2, it was with sensitivity of 84.0% and specificity of 64.0% with AUC of 0.80 (p <0.001, 95% C.I: 0.71 – 0.89). While for PIWIL3, it was with sensitivity of 68.0% and specificity of 84.0% with AUC of 0.79 (p < 0.001, 95% C.I: 0.70 – 0.88) and finally statistical significance was observed for PIWIL4, with sensitivity of 64.0% and specificity of 68.0% with AUC of 0.66 (p < 0.01, 95% C.I: 0.56 – 0.77), the above results showed that the 4 studied PIWIs RNA can be used to differentiate between tumour and non-tumour tissue samples (Table 3). A combined ROC curve was prepared to show an inclusive diagnostic performance appraisal, serum had an overall sensitivity and specificity of 100%, and an AUC of 1.0. Tissue samples exhibited sensitivity of 70.7%, specificity of 68%, and AUC of 0.733. Both of which were significant p<0.001. AFP was measured using ELISA, the values had sensitivity of 84%, Specificity of 64% with AUC of 0.828(p <0.001**,95% C.I:0.746, 0.910).

Analysis of the association between the tumour grade, and pattern and gene expression of the studied PIWIs RNA:

Regarding serum samples, no significant association was observed between the expression and the tumour grade of the patients. As for tissue samples, a significant association was observed with p<0.05 for PIWIL1 and patients diagnosed grade II versus (Vs) grade I, PIWIL4, had an association between the expression in tissue and patients diagnosed with grade III Vs grade I. p<0.05 (Supplementary Data (S.D): Table 5). In terms of the association between tumour pattern and PIWILs expression, no significant difference was detected in serum, while, in the tissue, PIWIL2 was significantly associated with patients p<0.05, with acinar/solid Vs solid with 10.9(9.9- 10.9) (S.D.: Table 6). Only PIWIL1 expression in serum was significantly associated with cirrhotic patients with 71.5(18.2- 128.2) and p<0.05 and no association was observed between PIWILs tissue expression and tumour stage neither in fibrotic nor cirrhotic patients (Supplementary Data: Table 7). Other parameters were investigated including associations between HAI, hepatomegaly, ascites, splenomegaly, oedema lower limbs, however no significant difference was detected (S.D.: Tables 8-12 respectively). The correlation of the biochemical parameters AST, ALT, Alb, Bilirubin, AFP, S-creatinine, in addition to the number of masses, tumour size, and steatosis were analysed versus gene expression results and no significant difference was observed (S.D.: Table 13).

HCC is estimated to be the result of a history of chronic liver disease (CLD) in 70%-90% of patients, with major causes being attributed to HCV or HBV infections ³. In Egypt, HCC is perceived as a particular health challenge, with an approximate two-fold rise in the number of cases, over the course of a decade ⁴. Currently, AFP is the only widely employed serological marker for screening for tumours, diagnosing HCC. However, serum AFP has a specificity of 76%- 94%, and a sensitivity of 39%- 65% for HCC; thus, hindering its clinical use ²². Ample efforts were placed into detecting higher performing biomarkers, AFP-L3, GP73, DCP, and GPC3, but to date, clinical performance was not outshone from AFP. Thus, a biomarker with potential for the diagnosis and detection of HCC at earlier stages is of imperative need.

In terms of the demographic data for the patients, the mean age was 57.2±8.1, 44% were females and 56% were males, which is typical for the risk factors associated with HCC. We found no association between the parameters, and expression of PIWIL. This was confirmed for HAI, hepatomegaly, ascites, splenomegaly, oedema lower limbs, number of masses. The aforementioned findings were in alignment with Taubert, et al., 2015, who observed similar outcomes for no association with clinicopathological features for colon cancer patients ²³, but was observed by Zhang, et al., and Li, et al., in breast and colon cancer respectively ^{24, 25}. For tumour grade, PIWIL1 & PIWIL4 had significant difference with tissue expression, which was confirmed in a study by Litwin, et al., for PIWIL1, & PIWIL2 in breast cancer ²⁶.

Recently, several reports have indicated that aberrant expression of PIWI at the mRNA and protein levels occurs in various types of tumours ^{27, 28}. PIWIL1/HIWI was previously linked with several types of cancers, with a pattern of overexpression, moreover, it was correlated with tumour grading and staging ^{29, 30}. Our findings confirmed the apparent role of PIWIL mRNAs for HCC, with a pattern observed of overexpression for all PIWIL isoforms. Previously, PIWIL2/HILI was identified as overly expressed in colorectal, prostate, breast, cervical, gastric and bladder cancer, all of which were determined to be overly expressed for both PIWIL1& PIWIL2 ²⁷. PIWIL3 & PIWIL4 was observed in renal cell carcinoma. The involvement of PIWI proteins was linked with multiple hallmarks of cancer including invasion, apoptosis evasion, metastasis and cell proliferation, as such they possess prospective diagnostic factors and biomarkers for cancer prognosis ^{17, 31}. Significant increased level of PIWIL1 was reported for colon, bladder, and hepatocellular carcinoma. Expression of the four members of the PIWI proteins was viewed as distinct in tumour tissue when compared with the adjacent non-tumorous tissue ^{12, 32}. PIWIL3 and PIWIL1 were assessed for expression levels by ³³ in colorectal cancer, and had non-significant expression statistically. Among all PIWIL genes, those assessed for expression and correlated with overall survival and recurrence-free survival were PIWIL3 & PIWIL4, in invasive urothelial bladder cancer ²⁶. A limitation of a study was not assessing the expression of PIWIL1 and PIWIL2 at the mRNA level, and conversely depending on Immunohistochemistry ³⁴, but they reported higher expression levels ³⁴. Research on the expression of PIWIL proteins has yet to be fully established. In the current study, expression of all PIWIL was assessed using real-time PCR method in HCC patients. The levels of expression of PIWIL1, PIWIL2, PIWIL3, PIWIL4 in tumour and nontumorous adjacent tissue was carried out in addition to serum samples from HCC patients and healthy controls, the expression was compared with clinical parameters. When compared to adjacent non-cancerous tissues, we found a significantly elevated expression for PIWIL1, PIWIL2, PIWIL3, &, PIWIL4 in HCC samples (p <0.001). These findings are consistent with prior findings in colorectal cancer, in which PIWIL1 mRNA levels in non-cancerous tissue were low or undetectable, but were dramatically raised in associated malignant tissue ³⁵. Additionally, they are confirmed in another study, where PIWIL1/HIWI was reported to have marked expression levels in HCC tissue, for patients who had undergone curative resection ³⁶. In breast cancer, PIWIL1 and PIWIL3 gene expressions were reported to be upregulated, whereas PIWIL2 and PIWIL4 were downregulated compared with normal breast tissue.

It is noteworthy to state that our research detected significant association between PIWIL1 & PIWIL4 expression with increasing tumour grade (p<0.05). Moreover, PIWIL1 in serum detected an association between tumour stage and its expression. PIWIL2 expression in tissue was also found to be associated with tumour pattern. These findings are similar to those of previous studies, which found elevated expression throughout the initial phases of cancer, implying the significant role of PIWIL-RNAs in cancer development ^{29, 31, 32, 37}.

In terms of diagnostic performance, the provision of non-invasive approaches for the diagnosis of HCC is of utter significance. Our findings showed that serum had an overall sensitivity and specificity of 100%, and an AUC of 1.0, meaning that they could be a powerful tool for the diagnosis and prognosis for HCC. Tissue samples exhibited sensitivity of 70%, specificity of 68%, and AUC of 0.733. Both of which were significant p<0.001. Although PIWIL3 and PIWIL4 were prognostic, PIWIL1 and PIWIL2 did not show significant impacts on survival (Krishnan, et al., 2016). Research on the usefulness of PIWIL and PiRNAs was determined with higher sensitivity e.g., 83.3% sensitivity and 89.3% specificity for colorectal cancer (CRC), in addition to varying percentages, all of which were higher than tissue is CRC patients ^38-41. For prognostic performance, and the determination of HCC risk logistic regression was done with odd ratios indicating statistical significance (p<0.001) in serum, and (p<0.05) in tissue, these results are in agreement with Mai, et al., with odd-ratio (OR) values for CRC cases diagnosed 1-3 years at 7.23, 2.80, 2.45, and 1.24, respectively ³⁹ and similar findings were also reported ⁴². Tosun et al., evaluated the predictive value of the serum expression level of PIWIL2 in prostate cancer, and was for similar outputs ⁴³. The reactivation of PIWI expression in cancer clearly suggests that these proteins are involved in the growth and differentiation of tumours ⁴⁴. According to our supplementary table 13, there was no clear correlation between the values of AFP and the expression of the PIWIL at the mRNA level for all markers, the literature indicates that, serum AFP has a specificity of 76%- 94%, and a sensitivity of 39%- 65% for HCC ²². This was similarly reported in our results for ELISA/AFP, which was significant (p<0.001), with sensitivity of 84%, Specificity of 64, this demonstrates a much higher performance for our studied markers for serum evaluation. Furthermore, Disruptions in PIWI-piRNAs pathway regulation has an effect on biological processes involving cancer progression e.g apoptosis, migration, proliferation and metastasis, which represents an indication that PIWI proteins suggests that PIWI proteins and piRNAs can be used as diagnostic biomarkers and therapeutic targets for the treatment of hepatocellular carcinoma⁴⁴, and this the manipulation of gene expression for PIWI and PiRNAs could lead to management in cancer progression, and an enhanced patient recovery.

In conclusion, our findings provide a novel perspective on the roles of PIWIL at the mRNA level in cancer development, and their promise of being diagnostic candidates for HCC disease progression. Differential protein expression in various cancer types could indicate reciprocal regulation between different piRNAs and PIWI genes. The epigenetic regulation of the observed variations in PIWIL1, PIWIL2, PIWIL3, PIWIL4 at the transcriptional and protein levels is an important problem that needs to be investigated further. Abnormal mRNA expression has substantial diagnostic value, and should be evaluated further for prognostic potential, and its effects on survival for HCC.

Funding details

The authors of this investigation declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability statement

The datasets generated during and/or analysed during the current study are available from the corresponding author or S.M. on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Research Institute office for IRB (NHTMRI-IRB) (Serial:2-2019).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

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Table 1: Demographics and Clinico-pathological characteristics for HCC patients

Clinico-pathological characteristics	Total number of patients N=50 (%)
Age (Mean±SD)	57.2±8.1
Sex Female	22 (44.0)
Male	28 (56.0)
ALT	61.4±15.5
AST	65.8±16.6
Alb	2.3±1.0
Bilirubin	2.9±1.1
AFP	75.0 (40.0- 150.0)
S-creatinine	0.9±0.4
No. of masses	1.1±0.2
Tumour size	2.25 (0.75- 4.25)
Tumour Grade I	29(38.7)
II	11 (14.7)
III	10 (13.3)
Pattern Acinar	30 (60.0)
Solid	17 (34.0)
Acinar/Solid	3 (6.0)
Steatosis	0.02 (0.02- 0.04)
Stage Fibrosis	8 (16.0)
Cirrhosis	42 (84.0)
HAI A1	15 (30.0)
A2	35 (70.0)
A3	0 (0.0)
Hepatomegaly Negative	41 (82.0)
Positive	9 (18.0)
Ascites Negative	30 (60.0)
Positive	20 (40.0)
Splenomegaly Negative	21 (42.0)
Positive	13 (26.0)
Oedema Lower Limbs Negative	37 (74.0)
Positive	29 (58.0)

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb) and alpha-fetoprotein (AFP). No. of masses are represented as Mean and SD. But Alpha feto-protein, Tumour size, and Steatosis (Fatty degeneration of hepatocytes (% of cells)) are represented as Median and Interquartile Range IQR (25% -75%). While Sex, Grade, Pattern, Stage, HAI (Hepatitis Activity Index (grade of hepatitis), Hepatomegaly, Ascites, Splenomegaly, and oedema Lower Limbs are represented as Frequency and percent.

Table 2: Logistic Regression Results for both tissue and serum samples of PIWIL mRNA in HCC patients

Serum groups	HCC Risk determination (Logistic Regression)
Serum groups	*OR (95% C. I)*	*p value*
PIWIL1	3.87(1.23 – 8.36)	<0.001**
PIWIL2	4.21(1.04 – 7.23)	<0.001**
PIWIL3	3.26(1.14 – 8.21)	<0.001**
PIWIL4	2.99(1.57 – 9.23)	<0.001**
Tissue groups	*OR (95% C. I)*	*p value*
PIWIL1	2.35(0.17 – 5.32)	0.01*
PIWIL2	2.21(0.15 – 4.24)	0.01*
PIWIL3	2.46(0.23 – 6.27)	0.01*
PIWIL4	1.65(0.19 – 3.14)	0.01*

The studied genes were represented as Median and Interquartile Range IQR (25% -75%); the Risk of HCC was conducted by logistic regression. * p<0.05, ** p<0.001. Fold change values were multiplied by 10⁵for approximation. OR: Odds ratio; CI: Confidence interval.

Table 3: ROC curve analysis for diagnostic performance for each of the studied PIWIL mRNAs in serum and tissue samples.

	Groups	Cut-off	Sensitivity	Specificity	AUC	95% C. I		p value
	Groups	Cut-off	Sensitivity	Specificity	AUC	Lower Bound	Upper Bound	p value
Serum	PIWIL1	<1.6	100.0	100.0	1.0	1.0	1.0	<0.001**
	PIWIL2	<0.56	100.0	100.0	1.0	1.0	1.0	<0.001**
	PIWIL3	<8.87	100.0	100.0	1.0	1.0	1.0	<0.001**
	PIWIL4	<1.02	100.0	100.0	1.0	1.0	1.0	<0.001**
	ELISA/ AFP	>0.97	84.0	64.0	0.828	0.746	0.910	<0.001**
Tissue	PIWIL1	<4.76	80.0	72.0	0.80	0.71	0.89	<0.001**
	PIWIL2	<3.69	84.0	64.0	0.80	0.71	0.89	<0.001**
	PIWIL3	<4.5	68.0	84.0	0.79	0.70	0.88	<0.001**
	PIWIL4	<0.94	64.0	68.0	0.66	0.56	0.77	<0.01*

Sn: Sensitivity, Sp: Specificity, AUC Area under curve and C.I: 95% Confidence Interval. * p<0.05, ** p<0.001.

Table 4: ROC curve analysis for diagnostic performance for the studied PIWIs RNA in serum and tissue samples overall.

Groups	Sensitivity	Specificity	AUC	95% C. I		*p value*
Groups	Sensitivity	Specificity	AUC	Lower Bound	Upper Bound	*p value*
Serum	100.0	100.0	1.0	1.0	1.0	<0.001**
Tissue	70.7	68.0	0.733	0.683	0.783	<0.001**

Sn: Sensitivity, Sp: Specificity, AUC Area under curve and C.I: 95% Confidence Interval. * p<0.05, ** p<0.001.

No competing interests reported.

SupplementaryData.docx

P- element Induced Wimpy Testis (PIWI), a Potential Candidate Marker for Hepatocellular Carcinoma

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Abstract

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Introduction

Materials & Methods

Results

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

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