WO2020124350A1

WO2020124350A1 - Prediction of hcv-related hcc recurrence by exosomal lnc-dancr levels

Info

Publication number: WO2020124350A1
Application number: PCT/CN2018/121708
Authority: WO
Inventors: Ming-Lung Yu; Shu-Chi Wang; Chia-Yen DAI; Wan-lung CHUANG
Original assignee: Kaohsiung Medical University
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2020-06-25

Abstract

Long non-coding DANCR (lnc-DANCR) as biomarker for the diagnosis, prognosis and monitor of cancer, including but not limited to hepatocellular carcinoma (HCC) is provided herein. In some embodiments, the method of evaluating the prognosis of hepatocellular carcinoma (HCC) in a subject comprises: isolating at least one transcript of lnc-DANCR from a biological sample of a subject; measuring the test expression level of the at least one transcript of lnc-DANCR; and evaluating the prognosis of hepatocellular carcinoma of the subject according to the comparison of the test expression level to a control expression level of the at least one transcript of lnc-DANCR. In some embodiments, a kit for evaluating the prognosis of hepatocellular carcinoma (HCC) in a subject comprises binding molecules to at least one transcript of lnc-DANCR, wherein the at least one transcript of lnc-DANCR is exosomal lnc-DANCR.

Description

PREDICTION OF HCV-RELATED HCC RECURRENCE BY EXOSOMAL LNC-DANCR LEVELS

FIELD OF INVENTION

The present invention is related to a method of evaluating cancer prognosis, in particular a method of evaluating hepatocellular carcinoma (HCC) using lnc-DANCR as a biomarker.

BACKGROUND OF INVENTION

Hepatitis C virus (HCV) infection is one of the major causes of cirrhosis and hepatocellular carcinoma (HCC) . It remains a threat for public health not only in Taiwan but also worldwide, with the World Health Organization (WHO) estimating between 3 and 4 million people worldwide being infected with hepatitis C virus (HCV) each year. HCV infection is associated with a 15-to 20-fold increase in risk for HCC compared with HCV-negative population in cross-sectional and case control studies. Most patients chronically infected with HCV are asymptomatic for many years, and the average time to develop HCC after onset of HCV infection is about 28 years.

It has been shown that liver cancer sometimes develops even after achieving viral eradication with Interferon-based Therapy (IFN-based therapy) . Thus, it is very important to determine factors responsible for HCC development in patients after going through such therapy, especially in the era of direct-acting antiviral agent (DAA) treatment. A high rate of tumor recurrence has recently been reported after the success of DAA treatment in patients with a prior history of treated HCC. Moreover, patients treated with DAA still maintained sustained virologic response (SVR) rates of over 90%, regardless of unfavorable conditions such as old age and severe fibrosis that give rise to a high risk of hepatocarcinogenesis. Therefore, it is crucial to identify risk factors for HCC development and to construct a post-treatment surveillance system in patients with HCC after viral eradication by DAA treatment. Consequently, prognostic markers and/or target molecules to help in preventing and managing HCV-related HCC are needed.

Long non-coding RNAs (lncRNAs) are non-protein coding transcripts with sizes greater than 200 nucleotides. Traditionally regarded as noise, they are now emerging as crucial to protein synthesis, transcriptional activation or repression, and have shown to affect processes such as DNA damage response, cell cycle and etc. Due to its importance in regulatory processes, studies have found that lncRNAs are associated with different types of cancers, including hepatocellular carcinoma (HCC) . To date, there have been few reports on differentially expressed lncRNAs in HCV-induced HCC. The field thus remains a large unexplored and undefined area that demands better therapeutic targets and more sensitive diagnostic markers.

DESCRIPTION OF INVENTION

Long non-coding DANCR (lnc-DANCR) as biomarker for the diagnosis, prognosis and monitor of cancer, including but not limited to hepatocellular carcinoma (HCC) is provided herein. The term “prognosis” provided in the present specification is defined as the diagnosing or predicting the occurrence of a certain disease, the prediction of the recurrence of a certain disease or the determination of the mortality rate and recurrence rate of a subject being diagnosed of a certain disease.

In some embodiments, a method of evaluating the prognosis of hepatocellular carcinoma HCC in a subject is provided. The method comprises the following steps: isolating at least one transcript of lnc-DANCR from a biological sample of a subject; measuring the test expression level of the at least one transcript of lnc-DANCR; and evaluating the prognosis of hepatocellular carcinoma of the subject according to the comparison of the test expression level to a control expression level of the at least one transcript of lnc-DANCR.

In some embodiments, the at least one transcript of lnc-DANCR isolated in the method of evaluating the prognosis of HCC in a subject is exosomal lnc-DANCR. The at least one transcript of lnc-DANCR of the method has a nucleotide sequence selected from a group consisting of SEQ ID NO: 1, 2, 3 and 4. In some embodiments, the biological sample used in the method is, blood, serum, plasma or at least one exosome. In some embodiments, the hepatocellular carcinoma evaluated is hepatitis C virus-related hepatocellular carcinoma. In some embodiments, the test expression level of the at least one transcript of lnc-DANCR is measured using quantitative real-time PCR (qPCR) , reverse transcriptase-polymerase chain reaction (RTPCR) methods, microarray, next-generation RNA sequencing, serial analysis of gene expression (SAGE) or immunoassays.

In some other embodiments, a method of treating HCC in a subject is provided. The method comprises the following steps: isolating at least one transcript of lnc-DANCR from a biological sample of a subject; measuring the test expression level of the at least one transcript of lnc-DANCR; evaluating the prognosis of hepatocellular carcinoma of the subject according to the comparison of the test expression level to a control expression level of the at least one transcript of lnc-DANCR; and treating the subject with surgical procedure, chemotherapy, radiation therapy or antiviral therapy if the subject is diagnosed with or determined with the recurrence of HCC.

In some embodiments, the at least one transcript of lnc-DANCR isolated in the method of treating HCC in a subject is exosomal lnc-DANCR. In some embodiments the at least one transcript of lnc-DANCR of the method has a nucleotide sequence selected from a group consisting of SEQ ID NO: 1, 2, 3 and 4. In some embodiments, the biological sample used in the method is, blood, serum, plasma or at least one exosome. In some embodiments, the hepatocellular carcinoma evaluated is hepatitis C virus-related hepatocellular carcinoma. In some embodiments, the test expression level of the at least one transcript of lnc-DANCR is measured using quantitative real-time PCR (qPCR) , reverse transcriptase-polymerase chain reaction (RTPCR) methods, microarray, next-generation RNA sequencing, serial analysis of gene expression (SAGE) or immunoassays.

In some embodiments, the surgical procedure of the treating method is Orthotopic liver transplantation (OLT) , percutaneous ethanol injection (PEI) , radiofrequency ablation (RFA) or selected transcatheter arterial chemoembolization (TACE) . In some embodiments, the radiation therapy is radioembolization or selective internal radiation therapy. In some embodiments, the chemotherapy of the method of treatment is targeted systemic chemotherapy where the agents used in the chemotherapy may block one or more steps in one or more pathways including Vascular endothelial growth factor (VEGF) , epidermal growth factor, Ras mitogen-activated protein kinase (MAPK) , insulin-like growth factor receptor, hepatocyte growth factor/c-MET, PI3K/PTEN/Akt/mammalian target of rapamycin (mTOR) and Wnt/β-Catenin pathways. In some embodiments, the antiviral therapy of the method of treatment is DAA treatment.

In some additional embodiments, a kit for evaluating the prognosis of hepatocellular carcinoma in a subject, which comprises binding molecules to at least one transcript of lnc-DANCR is provided. In some embodiments, the at least one transcript of lnc-DANCR that the molecules of the kit bind to is exosomal lnc-DANCR. In some embodiments, the at least one transcript of lnc-DANCR has a nucleotide sequence selected from a group consisting of SEQ ID NO: 1, 2, 3 and 4. In some embodiments, the hepatocellular carcinoma evaluated is hepatitis C virus-related hepatocellular carcinoma. In some embodiments, the binding molecules to at least one transcript of lnc-DANCR is used in biochips, microarrays, quantitative real-time PCR (qPCR) , reverse transcriptase-polymerase chain reaction (RTPCR) methods, next-generation RNA sequencing, serial analysis of gene expression (SAGE) or immunoassays.

BREIF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scatter chart comparing exosomal DANCR levels between HCV-related HCC patients with recurrence to non-recurrence patients.

FIG. 2 is a ROC analysis of exosomal DANCR expression level for HCV-HCC recurrence prediction.

FIG. 3 is a line chart of the cumulative rate of HCV-HCC recurrence in patients with high and low exosomes DANCR levels.

FIG. 4 is a line chart of the cumulative mortality rate of HCV-HCC patients after tumor surgery with high and low exosomes DANCR levels.

FIG. 5 is a set of triple repetition analysis of isolated exosomes size and number by NanoSight NS300.

FIG. 6 is a ROC analysis of Non-tumor DANCR expression level, Tumor DANCR expression level, Tumor/Non-Tumor (T/NT) DANCR expression level, Alpha-Feto Protien (AFP) expression level, and exosomal DANCR expression level for HCV-HCC recurrence prediction.

DETALED DESCRITION OF THE EMBODIMENTS

It should be understood that the detailed descriptions of the embodiments are to illustrate the preferred embodiments of the inventions and not to limit the present invention to certain embodiments. It should be noted that the invention is meant to cover all alternative embodiments that are within the same spirit and scope of the present invention.

Long non-coding DANCR (lnc-DANCR) as biomarker for the diagnosis, prognosis and monitor of cancer, including but not limited to hepatocellular carcinoma (HCC) is provided herein. In one preferred embodiment, DANCR expression level in circulating exosomes as a liquid biopsy was determined by first extracting total exosomes from 200uL of serum of the subject of interest by exosome precipitation solution. In some embodiments, plasma is used as the biologic sample to extract exosomes. Exosomes are preferred as a liquid biopsy due to its stability and ability to preserve for longer periods by freezing after extraction. In some embodiments, triple repetition analysis of isolated exosome size and number were carried out by NanoSight NS300 as illustrated in FIG. 5. DANCR q-PCR was then carried out by isolating total exosomal-RNA from the exosomes by Trizol reagent. Then cDNA was synthesized using 1 μg of the isolated RNA and a cDNA reverse transcription kit. Polymerase chain reactions (PCR) were performed in triplicate for each sample and analyzed using the ABI Prism 7900 fast detection system. The house-keeping genes were used to normalize the data before the amplified transcripts were analyzed by the threshold cycle (Ct) method using ABI 7900 system software. In a preferred embodiment, the DANCR primers used in the PCR has a nucleotide sequence selected from a group consisting of SEQ ID NO: 1, 2, 3 and 4.

Results show that exosomal DANCR expression levels correlate with HCC recurrence in HCV-HCC and circulating lnc-DANCR is associated with clinicopathological characteristics in HCV-related HCC recurrence and mortality. In FIG. 1, the scatter chart comparing exosomal DANCR levels between HCV-related HCC patients with recurrence to non-recurrence patients show that there is significant upregulated DANCR expression in the former compared to the latter (p<0.001*) .

FIG. 2 presents a ROC analysis of circulating exosomal DANCR for HCV-HCC recurrence prediction where the cutoff value for recurrence prediction by the ROC curve was dCt >-7.5 (lncDANCR-U6) . The area under the curve (AUC) is 0.83, with a sensitivity of 68.3 and a specificity of 85.7.

Further using Kaplan-Meier’s analysis revealed that 1-, 3-, and 5-y cumulative incidence of HCV-HCC recurrence rates were 43.3%, 75.6%, and 90.0%, respectively, for patients with higher circulating DANCR, compared to 20.2%, 34.8%, and 39.5%, respectively, among those of lower circulating DANCR (HR=3.80; CI: 2.52-5.84; p<0.001*) as indicated in FIG. 3 and in table 1 and table 2 below.

TABLE 1

HCC Recurrence in Low serum DANCR (dCT < -7.5, n=89)

Year	1	3	5	8
Cumulative no. of event	16	30	41	82
Cumulative no. at risk	73	59	48	7
Cumulative rate (%)	20.2	34.8	39.5	49.8

TABLE 2

HCC Recurrence in High serum DANCR (dCT > -7.5, n=90)

Year	1	3	5	8
Cumulative no. of event	35	67	81	88
Cumulative no. of at risk	55	23	9	2
Cumulative rate (%)	43.3	75.6	90.0	91.3

The 1-, 3-, and 5-y cumulative incidence of mortaliity were 4.4%, 22.4%, and 37.7%, respectively, for patients with higher circulating DANCR, compared to 3.4%, 12.5%, and 20.6%, respectively, among those of lower circulating DANCR (HR=2.20; CI: 1.29-3.89; p=0.004*) as in FIG. 4 and in tablle 3 and table 4 below.

TABLE 3

Mortality Rate in Low serum DANCR (dCT < -7.5, n=89)

Year	1	3	5	8
Cumulative no. of event	3	11	27	78
Cumulative no. of at risk	86	78	62	11
Cumulative rate (%)	3.4	12.5	20.6	31.6

TABLE 4

Mortality Rate in High serum DANCR (dCT > -7.5, n=90)

Year	1	3	5	8
Cumulative no. of event	3	21	38	80
Cumulative no. of at risk	87	69	52	10
Cumulative rate (%)	4.4	22.4	37.7	59.3

Additionally, as illustrated in table 5. below, mulltivariable Cox regression analysis revealed that the strongest predicative factors for HCV-HCC recurrence with Tumor/Adjacent Non-Tumor (T/ANT) DANCR ratio (Hazard Ratio (HR) =3.27; Confidence Interval (CI) : 1.92-6.03; p<0.0001*) and age >65 years (HR=1.47; CI: 1.00-2.17; p=0.05*) and cirrhosis (HR=1.53; CI: 1.05-2.25; p=0.03*) in Model 1. HR of recurrence was 2.34 (CI: 1.58-3.45; p<0.0001*) and 3.80 (CI: 2.52-5.84; p<0.0001*) for tumor DANCR with cirrhosis (HR=1.71; CI: 1.17-2.52; p=0.005*) in Model 2 and circulating DANCR with age >65 years (HR=1.67; CI: 1.12-2.49; p=0.011*) in Model 3, respectively. All of the DANCR for HR in Model 4 indicated that the circulating DANCR (HR=3.36; CI: 2.02-5.20; p<0.0001*) presented strongest predictive for HCV-HCC recurrence with T/NT DANCR ratio (HR=2.70; CI: 1.53-5.08; p=0.0004*) and older age (HR=1.64; CI: 1.00-2.45; p=0.015*) .

TABLE 5

TABLE 6

Also, as in Table 6 above, HR for HCV-HCC mortality in Model 4 also presented strongest predictive factor in circulating DANCR (HR=3.01; CI: 2.02-4.55; p<0.0001*) with Tumor/Non-Tumor (T/NT) DANCR ratio (HR=2.72; CI: 1.55-5.09; p=0.0004*) and older age (HR=1.64; CI: 1.10-2.45; p=0.02*) . HR of mortality for T/NT DANCR ratio, tumor DANCR, and circulating DANCR was 3.23 (CI: 1.50-8.43; p=0.0017*) , 2.20 (CI: 1.32-3.65; p=0.003*) with Pathology III/IV stage (HR=2.01; CI: 1.05-3.65; p=0.04*) , and 2.20 (CI: 1.29-3.89; p=0.004*) with Pathology III/IV stage (HR=2.02; CI: 1.05-3.69; p=0.046*) , respectively.

The diagnostic evaluation of hepatic and circulating DANCR for HCV-HCC recurrence was conducted by calculating the area under the ROC curve. As illustrated in FIG. 6. The results presented that the AUC of circulating DANCR was 0.831, which is higher than hepatic tissue DANCR expression level regardless of in-tumor-DANCR level (AUC=0.694) or in T/NT DANCR ratio (AUC=0.739) . The AUC of circulating DANCR is also significantly superior to that of AFP (AUC=0.419) , which has been a useful factor for HCC diagnosis.

TABLE 7

At the optimal cut-off values by ROC analysis in recurrence, the specificities for circulating and hepatic tumor DANCR were 85.7%and 91.2%, while the positive predictive value (PPV) were 91.3%and 91.5%, respectively as illustrated in Table 7 .

Collectively, results indicated that circulating exosomal lncRNA is a desirable liquid biopsy for prognostic biomarker of HCC recurrence after surgical resection for HCV-HCC.

Claims

A method of evaluating the prognosis of hepatocellular carcinoma (HCC) in a subject, comprising:

isolating at least one transcript of lnc-DANCR from a biological sample of a subject;

measuring the test expression level of the at least one transcript of lnc-DANCR; and

evaluating the prognosis of hepatocellular carcinoma of the subject according to the comparison of the test expression level to a control expression level of the at least one transcript of lnc-DANCR.
The method of claim 1, wherein the at least one transcript of lnc-DANCR is exosomal lnc-DANCR.
The method of claim 1, wherein the at least one transcript of lnc-DANCR has a nucleotide sequence selected from a group consisting of SEQ ID NO: 1, 2, 3 and 4.
The method of claim 1, wherein the biological sample is, blood, serum, plasma or at least one exosome.
The method of claim 1, wherein the hepatocellular carcinoma is hepatitis C virus-related hepatocellular carcinoma.
The method of claim 1, wherein the test expression level of the at least one transcript of lnc-DANCR is measured using quantitative real-time PCR (qPCR) , reverse transcriptase-polymerase chain reaction (RTPCR) methods, microarray, next-generation RNA sequencing, serial analysis of gene expression (SAGE) or immunoassays.
A kit for evaluating the prognosis of hepatocellular carcinoma in a subject, which comprises binding molecules to at least one transcript of lnc-DANCR.
The kit of claim 7, wherein the at least one transcript of lnc-DANCR is exosomal lnc-DANCR.
The kit of claim 7, wherein the at least one transcript of lnc-DANCR has a nucleotide sequence selected from a group consisting of SEQ ID NO: 1, 2, 3 and 4.
The kit of claim 7, wherein the hepatocellular carcinoma is hepatitis C virus-related hepatocellular carcinoma.
The kit of claim 7, wherein the binding molecules to at least one transcript of lnc-DANCR is used in biochips, microarrays, quantitative real-time PCR (qPCR) , reverse transcriptase-polymerase chain reaction (RTPCR) methods, next-generation RNA sequencing, serial analysis of gene expression (SAGE) or immunoassays.