KR101623145B1 - Composition for Detection of Small Hepatocellular Carcinoma and Hepatocellular Carcinoma Latent in a Cirrhotic Liver - Google Patents

Abstract

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

Description

Composition for Detection of Small Hepatocellular Carcinoma and Hepatocellular Carcinoma Latent in a Cirrhotic Liver}

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

Liver cancer, especially hepatocellular carcinoma (HCC), is the most common malignant tumor, the second and third most common cause of cancer death in China and Korea, respectively, and the sixth most common cancer worldwide, and the third cause of death among cancer patients. HCC is an important disease (El-Serag HB, Mason AC. N Engl J Med . 1999;340(10):745-750; Kazynski 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 the diagnosis of HCC include AFP, Lens culinaris agglutinin-reactive AFP (AFP-L3%) and a protein derived by 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 is known to have the ability to know the prognosis in advance (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. In addition, AFP may be elevated in patients with chronic liver disease without HCC. DCP measurement for HCC has a sensitivity of 48-62% and a specificity of 81-98%, but its sensitivity is only 15-30% in the case of 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 due to any cause causes 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; Fattovich G, et al. Gastroenterology. 2004;127:S35-850;Jang 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 divided and more experiments are needed to define their significance (Marrero JA, et al. Gastroenterology. 2009;137(1):110-118).

Therefore, a new biomarker is needed for initial diagnosis and to satisfy high-risk patients according to the risk of HCC occurrence.

As a related patent, Korean Patent Publication No. 1020090098490 is specific for the presence of a proteinaceous marker for early diagnosis of liver cancer, characterized in that it is one or a combination of two or more selected from the group consisting of the following polypeptides, and the amount, pattern, or both. A composition for diagnosis of liver cancer, characterized in that it contains a substance detected as: 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).

The present invention solves the above problems and has been conceived by the necessity of the above, 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 occurrence.

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

In addition, the present invention provides a composition for early detection of hepatocellular carcinoma (HCC) latent in liver cirrhosis, comprising an antibody specific to the peptide of LGIGQLTA QEVKSACYLR (SEQ ID NO: 1).

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

Hereinafter, the present invention will be described.

In the present invention, the present inventors 1) evaluate the clinical use of HCCR-1 in HCC and compare the performance of HCCR-1 with AFP and DCP tests, 2) prove its usefulness in the diagnosis of bovine HCC, and 3) the last. As a result, we investigated whether HCCR-1 is useful for early detection of HCC latent in cirrhosis.

Hereinafter, the present invention will be described in detail.

normal, CH , LC , And HCC Serum from AFP , DCP And HCCR -1 decision

As can be seen from FIG. 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, the serum level of HCCR-1 significantly increased from CH to LC group ( P <.0001) (Fig. 1, C). In contrast, there were no significant differences 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 non-malignant chronic liver diseases such as CH and LC (Fig. 1, C). In addition, serum HCCR-1 levels were significantly increased in LC compared to CH (Fig. 1, C). Therefore, it indicates that the serum HCCR-1 level gradually increases as the liver disease progresses.

Using the currently recommended cutoff AFP , DCP , And HCCR -1 clinical performance

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 the previous paper (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 those for normal individuals, CH, and LC ( P <.0001).

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

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

The sensitivity of HCC with higher serum levels of AFP, DCP, and HCCR-1 than normal values was analyzed to determine its usefulness for detecting early HCC according to tumor size. At 11 ng/mL cutoff, positive AFP was found in 63.8% of HCCs measured with diameter = 2 cm (Table 3). However, at the 200 ng/mL cutoff, the positive AFP was only 35.1%. Meanwhile, DCP and HCCR-1 were performed in large HCC with positive rates of 42.2% and 36.4%, respectively. Thus, the positive rates of AFP and DCP are higher than that of HCCR-1 among the 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 HCC with diameters <2 cm or less, 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 the highest at 63.4% in bovine HCC, it has only an 11 ng/mL cutoff, which also shows a high false positive rate in patients with non-malignant chronic liver disease. Therefore, AFP is more effective in detecting large HCC at 20 and 200 ng/mL cutoff levels ( P = .0010 and P <.0001, respectively). In contrast, at the 40 mAU/mL cutoff, DCP only detected 11.5% bovine HCC, while it was more effective in detecting large HCCs ( P <.0001). Therefore, this result suggests that DCP can be increased most in large HCCs (Table 3).

Accuracy, sensitivity, specificity, and positive of AFP , DCP and HCCR -1 in HCC Predictive Value (PPV), Negative Predictive Value (NPV)

In order to determine the optimal cutoff level that balances the false positive and false negative rates with the highest PPV, ROC analysis was performed for AFP, DCP, and HCCR-1, respectively. As shown in Figure 2A, the AUC (0.791, 95% CI: 0.769-0.813) for AFP in HCC was the highest 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 bovine HCC group (< 2 cm), the AUC for AFP and HCCR-1 were approximately the same as 0.663 (95% CI: 0.623-0.703) and 0.656 (95% CI: 0.604-0.708), respectively ( Fig. 2, B). These values are higher than that of DCP with an AUC of 0.514 (95% CI: 0.486-0.563). However, AFP and HCCR-1 are almost equivalent in several patients with bovine HCC derived from patients with non-malignant liver disease (Fig. 2, B). At the 10 ng/mL cutoff, AFP exhibits maximum sensitivity and specificity to differentiate between HCC-free and HCC-bearing patients. 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, that of DCP was 44.3%, 86.7%, and 58.7%, and that of HCCR-1, respectively, 41.6%, 87.4%, and 58.6% (Table 4).

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

Dormant in cirrhosis HCC In the initial detection of HCCR -One

The present inventors evaluated the clinical usefulness of HCCR-1 as a predictor of latent HCC outbreak among patients with cirrhosis confirmed to have HBV or HCV. Screening for HCC was performed after or by ultrasound, 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 onset of HCC. HCC was initially diagnosed by the radiological method. When the diagnosis of HCC is unclear, ultrasound guided tumor biopsy was performed and pathological diagnosis was made based on the Edmondson-Steiner Criteria. Patients with cirrhosis were classified 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 levels of 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 ). Conversely, HCC occurred in 6 of 40 LC patients with an increase in AFP levels of 20 ng/mL or more within 1 year (1-year cumulative risk, 15.0%) (FIG. 3 ). On the other hand, among LC patients with normal levels of HCCR-1 and AFP, the incidence of HCC was only 2.8% within 1 year. Although the HCC induction rates were not significantly different between HCCR-1-positive and AFP-positive LC patients ( P = .445), the HCC induction rates in HCCR-1 and AFP-positive LC patients were both AFP and HCCR-1. Was higher than in the LC patients negative for ( P <.0001) (FIG. 3 ).

Thus, the present invention 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 could be used as an indicator of the risk of developing HCC in the background of liver cirrhosis.

HCCR Preparation of an antibody against -1 and HCCR From -1 Epitope location

Recombinant HCCR-1 protein was used as an immunogen for polyclonal and mAb generation. Polyclonal antisera and I87 mAb against 167 to 360 amino acids of HCCR-1 were prepared. The area of binding to HCCR-1 for I87 mAb was determined using mass spectrometry analysis of the time-dependent trypsinized fragment pattern of HCCR-1. The mass spectrum was shown at each time point (Fig. 4, A). The epitope area was determined using mass spectrometry analysis of the time dependent trypsinized fragment pattern of HCCR-1. The epitope site of HCCR-1 recognized by the I87 mAb is LGIGQLTA QEVKSACYLR. The secondary structure of the HCCR-1 C-terminal amino acid and its binding area with I87 mAb are shown in Fig. 4B.

Table 1 is a table showing the demographics of the study group.

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

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

Table 4 is a table of 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 is used for detection of latent HCC in patients with cirrhosis, detection of small HCC, and diagnosis of both AFP- and DCP-negative HCC. I could see that I could.

1 shows the serum concentrations of AFP, DCP, and HCCR-1 in patients and healthy individuals with CH, LC, and HCC. Serum levels were checked 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 value. The vertical line represents the nearest value to the median ±1.5 x interquartile range (IQR), excluding outer values represented by separate points. The significance of the difference between the median values for different patient groups was determined using the Mann-Whitney test, and P values are shown.
FIG. 2 is a diagram showing ROC curves comparing AFP, DCP and HCCR-1 in individuals with non-malignant 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. The area under the ROC curve indicates that it has 95% confidence. AFP is green, DCP is blue, and HCCR-1 is brown.
3 is a diagram showing the cumulative incidence of HCC in patients with cirrhosis according to AFP and HCCR-1 levels. The 1-year cumulative incidence was 20.4% in cirrhosis patients with HCCR-1 levels of 10 ng/mL or higher, 15.0% in cirrhosis patients with AFP levels of 20 ng/mL or higher, and 2.6 in cirrhosis patients with normal HCCR-1 and AFP. %.
Figure 4 is a diagram showing the epitope mapping analysis of HCCR-1. a shows epitope mapping and MALDI-TOF analysis by acetylation. b shows the secondary structure of the HCCR-1 C-terminal amino acid and mAb I87 and its binding site.

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

The present invention was approved by the Institutional Review Board at each research center. The blood samples obtained were 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 patients with liver disease were recruited from January 2004 to February 2010 from the Catholic University of Korea, Nanjing University in China, and Peking University in 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 individuals with no history of liver disease and with normal biochemical values. 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 decompensated liver disease.

If histological information was not available, clinically diagnosed LCs were defined as follows: (1) platelet count <100,000/μL and including a brunted nodular liver edge accompanied by a spleen (>12 cm). Suggestive Ultrasound Results of Liver Cirrhosis; (2) esophageal or gastric varicose veins; Or (3) complications of cirrhosis of the liver including ascites, varicose 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 guidelines of the American Association for the study of liver disease (Bruix J, Sherman M. Hepatology. 2005; 42:1208-1236). The last training day was in June 2011. HCC ( n = 612) consisted of independent patients with HCC demonstrated histologically or radiographically. When the diagnosis of HCC was unclear, an ultrasound guided tumor biopsy was performed and a pathologic diagnosis was made based on the Edmondson-Steiner criteria.

Example 1: Serum HCCR -One, AFP , And DCP density

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 documents (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), and three other cutoff values for AFP were used. The DCP level was measured by an 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. The level of HCCR-1 was measured by a sandwich ELISA assay using rabbit polyclonal anti-HCCR-1 _167-380 serum and mouse anti-HCCR-1 _{167-380 I87 monoclonal antibody (mAb).} HCCR-1 of 10 ng/mL was used as a cutoff for diagnostic evaluation.

The calibration samples to build the calibration curve 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 is composed of Tween/PBS. The absorbance value (OD450) was converted to concentration using the 4-factor equation of the SOFTmax Pro version. Typical inter-assay variance for this test is 6.8% to 13.4%. The minimum limit of detection of the assay for HCCR-1 is 1.45 ng.ml.

Example 2: Antibody generation

The C-terminus of human HCCR-1 (GenBank accession no. AF 195651) cDNA encoding a polypeptide derived from 167-360 amino acid residues was cloned into a 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 to generate polyclonal antibodies (Yoon SK, Lim NK, Ha SA, Park YG, Choi JY, Chung KW, et al. Cancer. Res . 2004;64:5434-5441). For the production of monoclonal antibodies (mAb), BALB/c mice were immunized twice by 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 displaying the highest antibody titer received an _{HCCR-1 167-360 boost prior to spleen removal.} The splenocytes were fused with P3-X63-Ag8 myeloma cells.

Example 3: time dependent Tryptic By analysis of the intercept pattern Epitope Mapping

Maltose-binding protein (MBP)-HCCR-1 (10 μM) and I87 mAb (5 μM) were mixed in 3 μl PBS, and only MBP-HCCR-1 of the same concentration was prepared in PBS. To start the treatment reaction by adding 8 μM trypsin to the sample pre-cultured for 30 minutes and to obtain each solution (0.5 μM) at a certain time to terminate the cleavage reaction, a matrix solution (a-cyano-4-hydroxycinnamic acid matrix) It was mixed with 0.5% trifluoroacetic acid) in 70% acetonitrile containing. The finished sample was directly loaded 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 to collect the spectrum of the treated sections. All collected spectra were obtained in positive-ion reflector mode. 500 shots, typically with 400 to 500 power, were added per spectrum. For the analysis of the spectrum, the theoretical trypsinized fragments of MBP-HCCR-1 were calculated by ProteinProspector (http://www.prospector.ucsf.edu).

Example 4: Statistical analysis

The chi-square test or Fisher exact probability test was used for the discrete variable, and the ANOVA or Kruskal-Wallis test was used for the continuous variable. The significance of the difference between the median values for different patient groups was determined using the Mann-Whitney test. A P value of <.05 was considered significant. The optimal cutoff value in the diagnosis of HCC for AFP, DCP, and HCCR-1 was determined by receiver operating characteristics (ROC) curve analysis. To compare the clinical usefulness 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 overall diagnostic value was expressed using area under the ROC. To evaluate the cumulative risk of HCC occurrence, Kaplan-Meier curve and log-rank test were used. All analyzes were performed using a SAS system for Windows V 9.1.

<110> KIM, HYUNKEE <120> Composition for Detection of Small Hepatocellular Carcinoma and Hepatocellular Carcinoma Latent in a Cirrhotic Liver <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 Leu Gly Ile Gly Gln Leu Thr Ala Gln Glu Val Lys Ser Ala Cys Tyr 1 5 10 15 Leu Arg

Claims (1)

The level of HCCR-1 and the level of alpha fetoprotein (AFP) were measured by measuring the amounts of HCCR-1 (human cervical cancer 1 protooncogene) protein and AFP (alpha fetoprotein) Providing information on the prediction of latent hepatocellular carcinoma (HCC) in patients with liver cirrhosis, which includes predicting that hepatocellular carcinoma will occur within 6 months to 1 year with 20ng / ml or more.

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