WO2015020242A1 - Composition for diagnosing small hepatocellular carcinoma and hepatocellular carcinoma latent in cirrhotic liver - Google Patents

Abstract

The present invention relates to a composition for diagnosing small hepatocellular carcinoma and hepatocellular carcinoma latent in a cirrhotic liver.

Description

Hepatocellular carcinoma diagnostic composition latent in bovine hepatocellular carcinoma and cirrhosis

The present invention relates to a composition for diagnosing hepatocellular carcinoma latent in bovine hepatocellular carcinoma and cirrhosis.

Liver cancer, in particular hepatocellular carcinoma (HCC), is the most common malignant tumor, the second and third most common cause of cancer deaths in China and Korea, the sixth most common cancer worldwide, and the third cause of cancer among patients, HCC is an important disease (El-Serag HB, Mason AC.N Engl J Med. 1999; 340 (10): 745-750; Kaczynski J, Oden A. N Engl J Med. 1999; 341 (6): 451-452; Wands JR.N Engl J Med. 2004; 351 (15): 1567-1570; El-Serag H, Rudolph K.Gastroenterology. 2007; 132: 2557-25766-9).

Clinically useful markers for HCC diagnosis include AFP, Lens culinaris agglutinin-reactive AFP (AFP-L3%) and proteins derived from vitamin K absence or antagonist-II (PIVKA-II), known as DCP. Among them, serum AFP is most widely used as a tumor marker for detecting patients with HCC and has been known to have a prognostic ability (Stefaniuk P, Cianciara J, Wiercinska-Drapalo A. World J Gastroenterol. 2010; 16; (4): 418-424).

However, AFP levels have weak sensitivity and specificity for HCC (Stefaniuk P, Cianciara J, Wiercinska-Drapalo A. World J Gastroenterol. 2010; 16 (4): 418-424; Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, et al. J Hepatol. 2001; 35: 421-430). Not all HCCs secrete AFP. AFP may also be elevated in patients with chronic liver disease without HCC. DCP measurements for HCC have a sensitivity of 48-62% and specificity of 81-98%, but its sensitivity is only 15-30% for early HCC (Liebman HA, Furie BC, Tong MJ, Blanchard RA, Lo). KJ, Lee SD, et al. N Engl J Med. 1984; 310 (22): 1427-1431; Weitz IC, Liebman HA. Hepatology. 1993; 18 : 990-997; Bruix J, Sherman M. Hepatology. 2005; 42: 1208-1236; Marrero JA, Feng Z, Wang Y, Nguyen MH, Befeler AS, Roberts LR, et al. Gastroenterology. 2009; 137 (1): 110-118).

LC from any cause can lead to HCC and can be considered a premalignant condition. In fact, most patients with HCC have underlying cirrhosis (Kim YS et al. J Korean Med Sci. 2003: 18: 833-841; Fattigot G, et al. Gastroenterology. 2004; 127: S35-850; Jan JW. Korean J Hepatol. 2009; S40-49)

The main control for this high risk group is early diagnosis of HCC to enable curative treatment. As surveillance tests, the value of current serum markers such as AFP and DCP is disaggregated and more experimentation is needed to define their significance (Marrero JA, et al. Gastroenterology. 2009; 137 (1): 110-118).

Therefore, new biomarkers are needed for initial diagnosis and to satisfy high risk patients with the risk of HCC outbreaks.

As a related patent, Korean Patent Publication No. 1020090098490 discloses the presence or absence of proteinaceous marker for early diagnosis of liver cancer and its amount, pattern, or both, characterized in that one or more combinations selected from the group consisting of the following polypeptides. Liver cancer diagnostic composition comprising a substance for detecting: ERLN2_HUMAN (ER lipid raft associated 2 isoform 1; NCBI GI: 6005721); NDUS3_HUMAN (NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa (NADH-coenzyme Q reductase); NCBI GI: 4758788); PSB9_HUMAN (proteasome beta 9 subunit isoform 2 proprotein; NCBI GI: 23110932); SET_HUMAN (SET translocation (myeloid leukemia-associated); NCBI GI: 145843637); SSRD_HUMAN (signal sequence receptor, delta; NCBI GI: 5454090); TGM2_HUMAN (transglutaminase 2 isoform a; NCBI GI: 39777597); UGPA_HUMAN (UTP-glucose-1-phosphate uridylyltransferase; NCBI GI: 2136353).

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and the above-mentioned necessity, and an object of the present invention is to provide a new diagnostic composition for satisfying high-risk patients according to their risk of initial diagnosis and HCC development.

In order to achieve the above object, the present invention provides a composition for diagnosing small hepatocellular carcinoma (HCC) comprising an antibody specific to the peptide of LGIGQLTA QEVKSACYLR (SEQ ID NO: 1).

In one embodiment of the invention, the composition preferably further comprises an alpha fetoprotein (AFP) protein or an antibody thereof, des-γ-carboxyprothrombin (DCP) protein or an antibody thereof, or a combination thereof, but is not limited thereto. .

In another aspect, the present invention provides a composition for the initial detection of hepatocellular carcinoma (HCC) potential for cirrhosis, including an antibody specific for the peptide of LGIGQLTA QEVKSACYLR (SEQ ID NO: 1).

In one embodiment of the invention, the composition preferably further comprises an alpha fetoprotein (AFP) protein or an antibody thereof, des-γ-carboxyprothrombin (DCP) protein or an antibody thereof, or a combination thereof, but is not limited thereto. .

Hereinafter, the present invention will be described.

In the present invention, we 1) evaluate the clinical use of HCCR-1 in HCC and compare the performance of ACR and DCP tests with HCCR-1, 2) demonstrate its usefulness in the diagnosis of bovine HCC, and 3) We investigated whether HCCR-1 is useful for the early detection of HCC latent in cirrhosis.

Hereinafter, the present invention will be described in detail.

Determination of Serum AFP, DCP and HCCR-1 in Normal, CH, LC, and HCC

As can be seen in Figure 1, the median (range) levels of AFP, DCP, and HCCR-1 in the serum of serum HCC patients were significantly increased compared to that of the serum of LC patients ( P <.0001). In particular, serum levels of HCCR-1 were significantly increased from CH to LC group ( P <.0001) (FIG. 1, C). In contrast, there was no significant difference in serum levels of AFP (FIG. 1, A) and DCP (FIG. 1, B) between the CH and LC groups. The median (range) levels of AFP in the CH and LC groups were 3.7 (0.5-1,082) and 3.8 (0.75-969) ng / mL, respectively ( P = 0.3121) (FIG. 1, A). The median (range) levels of DCP in the CH and LC groups were 16.5 (2-206) and 17 (1.12-4,320) mAU / mL, respectively ( P = 0.9938) (FIG. 1, B). These results indicate that serum HCCR-1 levels in HCC patients are significantly higher than in nonmalignant chronic liver disease such as CH and LC (FIG. 1, C). Serum HCCR-1 levels were also significantly increased in LC compared to CH (FIG. 1, C). Thus, serum HCCR-1 levels gradually increase with progression of liver disease.

Clinical performance of AFP, DCP, and HCCR-1 using currently recommended cutoffs

We compared the clinical performance of AFP (11, 20, and 200 ng / mL), DCP (40 mAU / mL), and HCCR-1 (10 ng / mL) using the currently recommended cutoffs.

The present inventors used three different cutoff values for AFP based on previous papers (Gupta S, Bent S, Kohlwes J. Ann Intern Med. 2003; 139: 46-50; Trevisani F, D'Intino PE, Morselli -Labate AM, Mazzella G, Accogli E, Caraceni P, et al. J Hepatol. 2001; 34 (4): 570-575). Using this current cutoff value, at 11 ng / mL cutoff, AFP had the best performance with the highest rate for positive HCC (Table 2). Nevertheless, the positive rates of AFP, DCP, and HCCR-1 for the HCC group were all significantly higher than for normal subjects, CH, and LC ( P <.0001).

Although at 11 ng / mL cutoff AFP showed 63.7% for positive HCC, non-malignant groups such as normal individuals, CH, and LC showed maximum positive rates of 15.3%, 23.1%, and 18.3% for, respectively. (Table 2). In contrast, 200 ng / mL of AFP was performed at a positive rate of 28.8% for HCC.

On the other hand, positive DCP was found in 35.6% of HCCs while only 7.4%, 7.5%, and 7.6% in the normal, CH, and LC groups, respectively (Table 2). This suggests that DCP is more specific for HCC. Similarly, HCCR-1 was performed at a positive rate of 39.7% for HCC, while it was only 5.5% and 5.2% in normal subjects and CH, group, respectively (Table 2 ). Thus, diagnostic performance of DCP and HCCR-1 was similar in normal individuals and CH groups. However, the positive rate of HCCR-1 in the LC group was higher than that of DCP. This is consistent with the data in FIG. 1 showing that serum HCCR-1 levels increase significantly in LC.

The sensitivity of HCCs with serum levels of AFP, DCP, and HCCR-1 higher than normal values was analyzed to determine the utility for detecting early HCC depending on tumor size. Positive AFP at 11 ng / mL cutoff was found in 63.8% of HCCs measured diameter = 2 cm (Table 3). However, at 200 ng / mL cutoff the positive AFP was only 35.1%. DCP and HCCR-1, on the other hand, were performed with positive rates of 42.2% and 36.4%, respectively, in large HCCs. Therefore, the positive rates of AFP and DCP are higher than those of HCCR-1 between large HCCs with these currently recommended cutoffs (Table 3). However, HCCR-1 (10 ng / mL) was performed with a positive rate of 51.9% for small HCCs with diameter <2 cm, which is higher than positive AFP and DCP (Table 3).

In addition, positive HCCR-1 for bovine HCC is higher than for large HCC ( P = .0013). Although positive AFP is highest at 63.4% in bovine HCC, it has only 11 ng / mL cutoffs, which also show high positive rates in patients with nonmalignant chronic liver disease. Thus, AFP is more effective at detecting large HCCs at 20 and 200 ng / mL cutoff levels ( P = .0010 and P <.0001, respectively). In contrast, at 40 mAU / mL cutoff, DCP only detected 11.5% of small HCCs, while more effective in detecting large HCCs ( P <.0001). Thus, these results suggest that DCP can be most increased in large HCCs (Table 3).

Accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of AFP, DCP and HCCR-1 in HCC

ROC analysis was performed on AFP, DCP, and HCCR-1, respectively, to determine the optimal cutoff level that balanced the positive and negative rates with the highest PPV. As shown in FIG. 2A, AUC (0.791, 95% CI: 0.769-0.813) for AFP in HCC was highest when compared to that of DCP and HCCR-1 (0.678, 95% CI: 0.650-0.705 and 0.643, respectively). , 95% CI: 0.614-0.671). However, when evaluating only the small HCC group (<2 cm), the AUCs for AFP and HCCR-1 were approximately equal to 0.663 (95% CI: 0.623-0.703) and 0.656 (95% CI: 0.604-0.708), respectively ( 2, B). These values are higher than those of DCP with an AUC of 0.514 (95% CI: 0.486-0.563). However, AFP and HCCR-1 are nearly equivalent in several patients with bovine HCC derived from patients with nonmalignant liver disease (Figure 2, B). At 10 ng / mL cutoff, AFP exhibits maximum sensitivity and specificity to distinguish patients without HCC from patients with HCC. For DCP, the best cutoff level was 22 mAU / mL and for HCCR-1, the best cutoff level was 1.96 ng / mL. Based on this ROC defined cutoff, the sensitivity, specificity and PPV of AFP were 65.4%, 80.2%, and 58.6%, respectively, 44.3%, 86.7%, and 58.7% for DCP, and for HCCR-1, respectively. 41.6%, 87.4%, and 58.6% (Table 4).

When combined AFP, DCP, and HCCR-1, AUC improves from 0.891 (95% CI: 0.872 to 0.910) at 0.791 (AFP only), 0.678 (DCP only), and 0.643 (HCCR-1 alone) ( Data not shown). The combination of AFP, DCP, and HCCR-1 improves the sensitivity to HCC to 75.4% when using a cutoff that maximizes sensitivity + specificity (Table 4).

HCCR-1 in the Early Detection of HCC Latent in Cirrhosis

We assessed the clinical utility of HCCR-1 as a predictor of latent HCC incidence among patients with cirrhosis identified as having HBV or HCV. Screened for HCC post or by ultrasonography, CT, or MRI (Table 1). 19 LC patients were not performed. The remaining 589 patients with LC were included in this study for Kaplan-Meier analysis and performed every 3 or 6 months up to 12 months for the occurrence of HCC. HCC was initially diagnosed by a radiological method. If the diagnosis of HCC is unclear, an ultrasound guided tumor biopsy was performed and a pathological diagnosis was made based on Edmondson-Steiner Criteria. Liver cirrhosis patients were divided into three groups according to the levels of AFP and HCCR-1; 40 had AFP levels> 20 ng / mL, 93 had HCCR-1 levels> 10 ng / mL, and the remaining 456 had normal AFP and HCCR-1. HCC occurred in 19 of these 93 HCCR-1 positive LC patients within 1 year (1-year cumulative risk, 20.4%) (FIG. 3). In contrast, HCC occurred in 6 of 40 LC patients who had elevated AFP levels above 20 ng / mL within 1 year (1-year cumulative risk, 15.0%) (FIG. 3). In contrast, the incidence of HCC among LC patients with normal levels of HCCR-1 and AFP was only 2.8% within one year. Although the incidence of HCC was not significantly different between HCCR-1-positive and AFP-positive LC patients ( P = .445), the incidence of HCC in both HCCR-1 and AFP-positive LC patients was lower in both AFP and HCCR-1. Was higher than in patients with negative LC ( P <.0001) (FIG. 3).

The present invention thus suggests an association between the risk of HCC development and serum HCCR-1 levels in LC patients with elevated HCCR-1 levels. This suggests that HCCR-1 can be used as an indicator of the risk of HCC development in cirrhosis background.

Preparation of Antibodies to HCCR-1 and Location of Epitopes in HCCR-1

Recombinant HCCR-1 protein was used as an immunogen for polyclonal and mAb production. I87 mAbs for 360 amino acids at 167 of polyclonal antiserum and HCCR-1 were prepared. The binding area for HCCR-1 for the I87 mAb was determined using mass spectrometry analysis of the time dependent trypsin fragment pattern of HCCR-1. Mass spectra were shown at each time point (FIG. 4). Epitope areas were determined using mass spectrometry analysis of the time dependent trypsin section pattern of HCCR-1. The epitope region of HCCR-1 recognized by I87 mAb is LGIGQLTA QEVKSACYLR. The secondary structure of HCCR-1 C-terminal amino acid and its binding area with I87 mAb are shown in FIG. 5.

Table 1

Table 1 shows a demographic of the study group.

TABLE 2

Table 2 is a comparative table of AFP, DCP, and HCCR-1 in the study group.

TABLE 3

Table 3 is a comparative table of AFP, DCP, and HCCR-1 in the difference of patients with HCC according to tumor size.

Table 4

Table 4 is a table of the accuracy, sensitivity, specificity, PPV and NPV of AFP, DCP, and HCCR-1 in combination.

As can be seen from the present invention, the antibody of the present invention against HCCR-1 can be used for the detection of HCC latent in liver cirrhosis patients, the detection of small HCC, and the diagnosis of both AFP- and DCP-negative HCC. Could see.

1 shows serum concentrations of AFP, DCP, and HCCR-1 in patients with CH, LC, and HCC and in healthy individuals. Serum levels examined by ELISA. Box plots for AFP (Panel A), DCP (Panel B), and HCCR-1 (Panel C) levels in four groups of individuals. The boxes represent the 25 ^th and 75 ^th percentiles of the data and the middle line represents the median. The vertical line represents the value nearest to the median ± 1.5 x quartile range (IQR), excluding outside values represented by discrete points. The significance of the difference between the median values for the various patient groups was determined using the Mann-Whitney test, indicating P values.

FIG. 2 shows ROC curves comparing AFP, DCP and HCCR-1 in individuals with nonmalignant liver disease versus patients with HCC (Panel A) or Bovine HCC (Panel B). The curve shows an optimal cutoff value for AFP of 10 ng / mL, an optimal cutoff value for DCP of 22 mAU / mL, and an optimal cutoff value for HCCR-1 of 1.96 ng / mL. Area under the ROC curve indicates that it has 95% confidence. AFP is green, DCP is blue, and HCCR-1 is brown.

Figure 3 shows the cumulative occurrence of HCC in patients with cirrhosis according to AFP and HCCR-1 levels. The 1-year cumulative incidence was 20.4% in patients with cirrhosis with HCCR-1 levels above 10 ng / mL, 15.0% in patients with cirrhosis with AFP levels above 20 ng / mL, and 2.6 in patients with cirrhosis who were normal for both HCCR-1 and AFP. %.

Figure 4-5 shows epitope mapping analysis of HCCR-1. 4 shows epitope mapping and MALDI-TOF analysis by acetylation. Figure 5 shows the secondary structure of HCCR-1 C-terminal amino acid and mAb I87 and its binding site.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.

The present invention was approved by the Institutional Review Board at each research center. The obtained blood sample was given the written consent of all patients. The use of blood samples was approved by the ethics committee of each clinical trial committee.

418 normal and 1622 liver disease patients were recruited from January 2004 to February 2010 from the Catholic University of Korea, Nanjing University of China and Peking University of China (Table 1).

The liver disease patient cohort ( n = 1622) consisted of 402 CH patients, 608 LC patients, and 612 HCC patients. Normal ( n = 418) included normal healthy subjects with no history of liver disease and with normal biochemistry. CH ( n = 402) consisted of patients with histologically confirmed non-cirrhosis chronic hepatitis. LC ( n = 608) included patients with histologically confirmed cirrhosis with compensated or non-target liver disease.

If histological information was not available, clinically diagnosed LCs were defined as follows: (1) with a blunted nodular hepatic edge accompanied by platelet counts <100,000 / μL and splenomegaly (> 12 cm). Indicative Ultrasound Results of Liver Cirrhosis; (2) esophageal or gastric varicose veins; Or (3) complications of cirrhosis, including ascites, varicose vein bleeding and hepatic encephalopathy.

If no evidence of HCC was detected, patients performed AFP and ultrasonography every 3 or 6 months. During surveillance, HCC was diagnosed based on the American Association's guidelines for the study of liver disease (Bruix J, Sherman M. Hepatology. 2005; 42: 1208-1236). The last performance date was June 2011. HCC ( n = 612) consisted of independent patients with histologic or radiologically proven HCC. When the diagnosis of HCC was unclear, an ultrasound guided tumor biopsy was performed and a pathological diagnosis was made based on the Edmondson-Steiner criteria.

Example 1 Serum HCCR-1, AFP, and DCP Concentration

AFP levels were measured by enzyme immunoassay (EIA) (Elecsys Modular Analytics E170, Roche Diagnostics, Mannheim, Germany). We used three cutoffs (11, 20, or 200 ng / mL) for the determination of the positive reaction. The present inventors have previously published literature (Gupta S, Bent S, Kohlwes J. Ann Intern Med. 2003; 139: 46-50; Trevisani F, D'Intino PE, Morselli-Labate AM, Mazzella G, Accogli E, Caraceni P , three al cutoff values for AFP were used, based on et al. J Hepatol. 2001; 34 (4): 570-575). The DCP level was measured by enzyme immunoassay (EIA) (Sanko Junyaku Co., Ltd., Tokyo, Japan). To determine the positive response, we used a 40 mAU / mL value as the cutoff for DCP as directed by the manufacturer. Levels of HCCR-1 were measured by sandwich ELISA assays using rabbit polyclonal anti-HCCR-1 _167-380 serum and mouse anti-HCCR-1 _167-380 I87 monoclonal antibody (mAb). 10 ng / mL of HCCR-1 was used as the cutoff for diagnostic evaluation.

Calibrator samples to build calibration curves consisted of several concentrations of HCCR-1 diluted in 1% BSA and 0.1% Tween / PBS, and the negative control was 1% BSA and 0.1% without HCCR-1 protein. It consists of Tween / PBS. Absorbance values (OD450) were converted to concentrations using the 4-coefficient equation of the SOFTmax Pro version. The typical assay variation for this test is 6.8% to 13.4%. The minimum limit for detection of an assay for HCCR-1 is 1.45 ng.ml.

Example 2: Antibody Production

The C terminus of the human HCCR-1 (GenBank accession no. AF 195651) cDNA encoding a polypeptide derived from the 167-360 amino acid residue was cloned into the pMAL-p2X (New England Biolabs, Beverly, MA) vector. The recombinant HCCR-1 protein was purified using amylose resin. The fusion protein was digested with factor Xa protease, and the resulting HCCR-1 protein was used for polyclonal antibody production (Yoon SK, Lim NK, Ha SA, Park YG, Choi JY, Chung KW, et al. Cancer Res. 2004; 64: 5434-5441). For monoclonal antibody (mAb) production, BALB / c mice were immunized twice with sc injection. The first immunization was performed using 50 μg HCCR-1 _167-360 in Freund's complete adjuvant, and two weeks later a second immunization was started with HCCR-1 _167-360 . Mice showing the highest antibody titers received HCCR-1 _167-360 boost prior to spleen removal. The splenocytes were fused with P3-X63-Ag8 myeloma cells.

Example 3: Epitope Mapping by Analysis of Time-dependent Tryptic Section Patterns

Maltose-binding protein (MBP) -HCCR-1 (10 μM) and I87 mAb (5 μM) were mixed in 3 μl PBS and only the same concentration of MBP-HCCR-1 was prepared in PBS. Add 8 μM trypsin to the preincubated sample for 30 minutes to start the treatment reaction and obtain each solution (0.5 μM) at any specific time to terminate the cleavage reaction (a-cyano-4-hydroxycinnamic acid matrix 0.5% trifluoroacetic acid in acetonitrile containing 70%). The finished sample was loaded directly onto a matrix-assisted laser desorption / ionization plate or kept at −20 ° C. until further analysis. Ettan Pro matrix-assisted laser desorption / ionization time-of-flight (Amersham Biosciences, Piscataway, NJ) was used for spectral collection of treated sections. All collected spectra were obtained in positive-ion reflector mode. Typically 500 shots with 400 to 500 power were added per spectrum. For the analysis of the spectra, theoretical trypsin sections of MBP-HCCR-1 were calculated by ProteinProspector ( http://www.prospector.ucsf.edu ).

Example 4: Statistical Analysis

The chi-square test or the Fisher exact probability test was used for the discontinuous variables, and the ANOVA or Kruskal-Wallis test was used for the continuous variables. The significance of the differences between medians for the various patient groups was determined using the Mann-Whitney test. P values of <.05 were considered significant. Optimal cutoff values in the diagnosis of HCC for AFP, DCP, and HCCR-1 were determined by receiver operating characteristics (ROC) curve analysis. To compare the clinical utility of AFP, DCP, and HCCR-1, area under the ROC (AUC) was calculated and performed by comparing patients with HCC and non-HCC patients. The best cutoff values for AFP, DCP, and HCCR-1 were automatically selected with the highest diagnostic accuracy (ie, the sum of false-negative and false-positive rates was minimized). Each total diagnostic value is shown using area under the ROC. To assess the cumulative risk of HCC development, Kaplan-Meier curves and log-rank tests were used. All analyzes were performed using a SAS system for Windows V 9.1.

Claims (6)

1. A composition for diagnosing small hepatocellular carcinoma (HCC) comprising an antibody specific for the peptide sequence of LGIGQLTA QEVKSACYLR (SEQ ID NO: 1).
2. According to claim 1, wherein the composition is bovine hepatocellular carcinoma diagnostic composition, characterized in that it further comprises an AFP (alpha fetoprotein) protein or an antibody thereof.
3. According to claim 1, wherein the composition is bovine hepatocellular carcinoma diagnostic composition, characterized in that it further comprises a DCP (des-γ-carboxyprothrombin) protein or an antibody thereof.
4. LGIGQLTA QEVKSACYLR (SEQ ID NO: 1) A composition for early detection of hepatocellular carcinoma (HCC) latent in cirrhosis, including an antibody specific for the peptide sequence.
5. The composition for initial detection of hepatocellular carcinoma latent in cirrhosis, characterized in that the composition further comprises an AFP protein or an antibody thereof.
6. 5. The composition of claim 4, wherein the composition further comprises a DCP protein or an antibody thereof.

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