Major General Prof. Dr. LE TRUNG HAI

President, Vietnam Association for the Study of Liver Diseases

Dr. LE TRUNG HIEU

Department of Hepato-Biliary Pancreatic Surgery, CMH 108 at al

 

SUMMARY:

+ Aims: Evaluate validity of DKK1 and HBx-LINE1 in diagnosis and post-treatment of HCC/HBV and analyse relationship with clinical and paraclinical some characteristics.

+ Methods: Study on 114 HCC patients at Central Military Hospital (CMH) 108 and Military Hospital (MH) 103 (1/2016-3/2018). DKK1 and HBx-LINE1 were analyzed in Department of Microbiology of CMH 108, including using Realtime PCR technique with Taqman probe for level of mRNA DKK1 expression and ELISA technique for serum protein DKK1. Total RNA was transcribed to cDNA and subsequently amplified by hemi-nested PCR in order to identify HBx-LINE1 transcripts.

+ Results: With DKK1 ≥2.15 ng/mL, the positive rates of serum protein DKK1 were significantly increased when compared with those of AFP. The mean of serum protein DKK1 of HCC was significantly higher than it in liver cirrhosis (LC) patients with p<0.05. Combination between AFP and DKK1 expression will improved positive rates and help more diagnosis in 12.3% of HCC cases. Logistic regression analysis showed the risk of HCC will increased about 18.5 times when DKK1 ≥2.15 ng/mL. The level of DKK1 expression reduced in patients with ≥2 tumors when compare with one tumor and in cases with size ≥5 cm when compare with <5 cm. This correlation was the same with reducing of AFP related with quantity and size of tumor. The level of DKK1 expressions reduced in post-hepatectomy when compare with those before surgery and in time of more than one year when compare with those from 1 to 12 months. We did not identify HBx-LINE1 fusion transcript in all 114 (100%) HBV-related HCC patients. 

+ Conclusion: Biomarkers serum protein DKK1 and DKK1 expression have validity in diagnosis and post-treatment monitoring of HCC, especially for AFP-negative patient. HBx-LINE1 fusion transcript was not identified in our study. 

INTRODUCTION

Hepatocellular carcinoma (HCC) - now the most common cancer in Vietnam with mortality about more than 25 000 cases per year (Globocan 2018)(4). HBV infection is a top risk for HCC in Vietnam with about 60-80% HCC patients associated HBV(+). Study of TN Dung (2018) (1) in 3 biggest military hospitals showed HCC was also the number one. Diagnosis of HCC based on three main factors: imaging diagnosis, biomarker tests and histo-pathology. AFP is a biomarker with sensitive rate about 60% when level >20 ng/mL. So that about 40% of AFP was false negative. AFP-L3 and DCP helped to increased diagnosis rate. When combined them will gain detection of HCC, especially for tumor less than 2 cm. But the cost of diagnosis and only of some medical centers can perform these tests. So that, we studied to find one new biomarker to increase sensitive, specification and support for AFP also for small tumor in diagnosis, prognosis treatment effectiveness monitoring of HCC.

Dickkopf-1 (DKK1), a secretory antagonist of the canonical Wnt signalling pathway, was identified in 1998. It is hardly expressed in normal human adult tissues, except in placental and embryonic tissues. The large-scale, multicenter study of Shen at al (2012) (6) showed that serum DKK1 could potentially be used to diagnose HCC, especially early-stage disease, and will help to resolve the deficiencies of AFP in the testing of AFP-negative patients, and can be used to make differential diagnoses. Other study of Jie Zhang (2014) (3) also indicated DKK1 or DKK1 combined with AFP help exact diagnosis of HCC with high level.

HBx-LINE1 - a HBV-human chimeric fusion transcript was described in 2014 (4) and its has functions as a long noncoding RNA and associated with tumor formation hepatitis B virus (HBV)-induced liver carcinogenesis. Study in Hongkong by Lau et al (7) showed that HBx-LINE1 was occurred in 23.3% of HBV-associated HCC tumors and associated with poor prognosis in patients with HCC from Hongkong. Jessica Zucman-Rossi (2014) (12) did not identify HBx-LINE1 fusion transcript in a series of 50 HBV-related HCC from Europe. He discussed that the result of Lau et al could be restricted to the Hong Kong population where HBV genotype C is predominant and need other study to evaluate.

In Vietnam there are still not any study about biomarkers DKK1 & HBx-LINE1. Our study to evaluate validity of DKK1 and HBx-LINE1 in diagnosis and post-treatment monitoring of HCC/HBV and analyse relationship with clinical and paraclinical some characteristics.

METHODS

          Study on 114 HCC patients and 6 LC patients at CMH 108 and MH 103 (1/2016-3/2018). DKK1 and HBx-LINE1 were analyzed in Department of Microbiology of CMH 108, including using Realtime PCR technique with Taqman probe for level of mRNA DKK1 expression and ELISA technique (with kit from R&D Systems, Inc) for serum protein DKK1. For identifying of HBx-LINE1 transcripts: the quality of total RNA preparations was assessed by NanoDrop spectrometer. Approximately 500 ng of total RNA were used for reverse transcription (RT) by RevertAid First Strand cDNA Synthesis Kit following the manufacturer’s instructions. Reverse-transcribed cDNA was subsequently amplified by hemi-nested PCR using the primer pairs as described by Lau et al. (7). A synthesized plasmid sequence as described containing the fusion HBx-LINE1 transcript was utilized as positive control. The GAPDH cDNA gene transcript (496bp) was used as an internal control. In both PCR reactions, the annealing temperatures were 60°C and products were amplified in 30 cycles. PCR products were analyzed on 1.5% agarose gel together with the positive plasmid control.        

RESULTS AND DISCUSSION 

* Result of DKK1 test

- Serum protein DKK1 concentration:

Table1. Serum protein DKK1.

Serum protein DKK1 (ng/mL)	Hepatectomy for HCC group		Biopsy for HCC group		Both
	N	%	N	%	N	%
<2,15	2	6.06	1	1.23	3	2.63
	31	93.94	80	98.77	111	97.37
	33	100	81	100	114	100
Mean (min-max)	133.3± 94.48 (0-336.39)		123.68±96.19 (0-456.02)		126.47±94.83 (0-456.02)

Table 2. Serum AFP

Serum AFP (ng/mL)	Hepatectomy for HCC Group		Biopsy for HCC Group		Both
	N=33	%	N=81	%	N=114	%
<20	16	48.48	25	30.86	41	39.96
≥20	17	51.52	56	69.14	73	60.04
Mean (min-max)	534.15±924.14 (0.85-3300)		734.76±894.05 (1.93-2479)		659.54±905.43 (0.85-3300)

Positive rate of serum protein DKK1 (97.37%) was significantly higher than positive rate of serum AFP (60.04%). Mean concentration of serum protein DKK1 of HCC was 126.47 ± 94.83 ng/mL significantly higher than that of liver cirrhosis (37.04 ± 33.96 ng/mL) with p<0.05.

Tung EK.K. et al 2012 (3), Prieto P.A. et al 2013 (9), J. Zhang et al 2014 (6), Y.M. Fouad et al 2016 (11): DKK1 additional for AFP in diagnosis of HCC, especially for the patient with AFP (-).

          - Gene expression of mRNA DKK1:

21.05% patients: gene expression DKK1 (+), including 12.3% patients with AFP (-). Combination of AFP and gene expression DKK1 improved (+) rate and more diagnosis in 12.3% of patients with HCC.

M. Watany at al 2017 (8) tested gene expression DKK1 in 50 HCC cases and 10 control cases showed DKK1 was one promising biomarker for diagnosis, even patient with AFP (-) and AFP associated with DKK1 improved accurate diagnosis of HCC/HBV.

* Relationship between DKK1 and some clinical and paraclinical characteristics:

- In hepatectomy group due to HCC: gene expression DKK1 reduced in tumor number ≥2 tumors when compared to 1 tumor. This reduction was the same when tumor size ≥5cm compared with <5 cm. Both above reduction were the same with AFP exchange about tumor number and size.

+ Combination with DKK1 in diagnosis when AFP (-) in hepatectomy group: in 16 patients with AFP (-) but serum protein DKK1 (+). So that, if using combination all 3 tests: AFP, serum protein DKK1 & gene expression mRNA DKK1 will improving more diagnosis for patient with AFP (-).

+ Logistic regression analysis

Table 3. Logistic regression analysis with serum protein DKK1 in HCC patients

Protein DKK1 (ng/mL)	HCC group		LC group		OR	95% (CI)	P
	N	%	N	%
<2.15	3	2.63	2	33.33	18.5	2.39- 143.49	p=0.005
≥ 215	111	97.37	4	66.67
Total	114	100.00	6

Logistic regression analysis showed risk factor of HCC in patient with serum protein DKK1 group ≥2.15 ng/mL higher 18.5 times when compared with liver cirrhosis group with p<0.05.

* Value for posttreatment monitoring of HCC and relationship of DKK1: from 33 cases of hepatectomy due to HCC: 19 cases came back for re-examination and DKK1 test. Mean time of re-examination 11.68 months (4-20 months).

- Result: change of gene expression DKK1 at post-treatment time (0.98±2.10) was reduced when compared to before surgery (2.23±6.49) and gene

expression DKK1 after surgery >12 months (0.22±0.09) was reduced when compared to before this point (1-12 months) (with gene expression DKK1 of 1.28±0.59).

          - Correlation between gene expression DKK1 level and AFP before and after hepatectomy: Post-surgery gene expression DKK1 level was reduced when compare to before surgery. Post-surgery AFP rate (-): 73.91%, AFP (+) 26.09%.

Jeng J-E at al. 2014 (5): gene expression DKK1 level was reduced after hepatectomy. Recent studies showed serum DKK1 level was prognostic marker when noninvasive HCC, especially for early HCC or HCC with AFP (-) HCC patient with high level of gene expression DKK1 were bad prognosis. That is new prognostic factor in HCC.

* Result of HBx-LINE1 test

In the 114 tumor and matched serum samples, HBx-LINE1 transcripts were not detected in any of the samples examined. The positive control remained positive & the negative control was negative in all assays.

These results were the same with those of the study of J. Zucman - Rossi at al. 2014 (France) (12) and one again showed that except result of C.C. Lau (Hongkong) detected HBx-LINE1 in 23.3% (a chimerical transcript recently identified in a Chinese population of HBV-related HCC was claimed to be associated with tumor formation and poor patient survival). Studies outside of Hongkong still not found any HBx-LINE1 transcript expression in HCC/HBV.

While ethnic variation might in part account for such a discrepancy. In Vietnam with high rate of HBV infection (15-20%), in that B type gene was most common (45.5% BH Hoang - 2004) (2) so different to those of Hongkong (where C type gene was predominant).

So that, at this moment, we must definitely conclude that, based on our results and the previous study of  Zucman-Rossi and others, any usefulness of the HBx-LINE1 transcript as a prognostic biomarker must clearly be rejected. Further studies are warranted to assess any prognostic potential of HBx-LINE1 in HBV-associated HCC development.

CONCLUSION

Study on 114 patients with HCC/HBV in two biggest military hospitals in Hanoi, Vietnam (1/2016-3/2018) showed that:

* With level ≥2.15 ng/mL, serum protein DKK1 (+) rate in groups hepatectomy, biopsy and both were significantly higher when compared with AFP (+) rate (with level ≥20 ng/mL). Serum protein DKK1 concentration in HCC group was significantly higher when compared with LC group (p<0.05).

- Using combination both AFP and gene expression mRNA DKK1 was improved (+) rate and helped more diagnoses in 12.3% HCC cases.

- Combination three tests AFP, serum protein DKK1 and gene expression DKK1 helped more diagnosis for HCC with AFP (-).

- Logistic analyze showed HCC risk in group, who had serum protein DKK1 ≥2.15 ng/mL higher 18.5 times when compared with LC group (p<0.05).

- Gene expression mRNA DKK1 reduced after hepatectomy and post-surgery >12 months. Gene expression mRNA DKK1 reduced when tumor number >2 and size ≥5cm, similar to AFP concentration.

* The HBx-LINE1 transcripts were not detected in any of our clinical samples. The confirmation of role of HBx-LINE1 as prognostic biomarker need to again evaluate and a high rate of this transcripts in HCC/HBV patients should more study to evaluate./.

 

References

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