CN112034171A - Application of reagent for detecting serum sphingosine-1-phosphate in preparation of kit for distinguishing liver cirrhosis or hepatocellular carcinoma - Google Patents
Application of reagent for detecting serum sphingosine-1-phosphate in preparation of kit for distinguishing liver cirrhosis or hepatocellular carcinoma Download PDFInfo
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Abstract
The embodiment of the invention relates to application of a reagent for detecting serum sphingosine-1-phosphate in preparing a kit for distinguishing liver cirrhosis or hepatocellular carcinoma. Up-regulation of serum Sph (d18:1) -1-P showed good diagnostic performance on HCC. In particular Sph (d18:1) -1-P can also be used as a biomarker for diagnosing AFP-negative HCC. These findings are helpful in the non-invasive diagnosis of HCC, including AFP-negative HCC.
Description
Technical Field
The invention relates to the field of biological detection, in particular to application of a reagent for detecting sphingosine-1-phosphate (Sph (d18:1) -1-P, S1P) in preparation of a kit for distinguishing liver cirrhosis or hepatocellular carcinoma.
Background
Hepatocellular carcinoma (HCC) is the third leading cause of death among cancer-related deaths in the world, with 70% to 90% of HCC likely coming from cirrhosis patients. Early discrimination of HCC from cirrhosis and further providing timely treatment can significantly improve the prognosis of HCC. However, there are no typical symptoms at the early stages of HCC and specific diagnostic biomarkers for HCC are missing, so many patients are already in late stages when diagnosed. This fact misses many patients the best opportunity for radical treatment, such as liver resection or liver transplantation.
To date, in clinical practice, imaging methods and alpha-fetoprotein (AFP) are commonly used to screen and diagnose HCC. On the one hand, however, ultrasound does not provide sufficient sensitivity and objectivity, possibly leading to diagnostic inaccuracies; computed Tomography (CT) and Magnetic Resonance Imaging (MRI) are not recommended as general screening tools for HCC because of radiation exposure or high cost; on the other hand, the positive rate of AFP is limited to only one-half to two-thirds, i.e. approximately 40% of patients with HCC cannot detect AFP. In addition, AFP may be affected by many non-HCC diseases (e.g. hepatitis, cirrhosis, cholangiocarcinoma, etc.). In summary, the current efficacy of serum biomarkers for the diagnosis of HCC, including AFP-negative HCC, is still not ideal. Therefore, there remains an urgent need to discover new biomarkers during the screening of HCC, in particular AFP negative HCC.
Sphingolipid metabolism is highly interconnected, wherein: ceramides occupy the core of the sphingolipid metabolic network, and can be glycosylated, phosphorylated, or deacylated to produce a wide variety of metabolites; wherein: sphingosine (Sph) can be produced by ceramidase and is further phosphorylated to sphingosine-1-phosphate by sphingosine kinase 1(SphK1) (see fig. 1).
The types and amounts of sphingolipids detected in prior studies are not comprehensive, e.g., certain carbon chain length ceramides or S1P are not included, which may lead to missing optimal diagnostic biomarkers. At the same time, the sensitivity and specificity of these individual markers was not given, nor was their diagnostic effect on AFP-negative HCC assessed. In addition, in view of the different etiologies and ethnicity, it is necessary to further screen for sphingolipid metabolites that can distinguish HCC (including AFP negative HCC) due to various causes from cirrhosis in chinese population with high sensitivity and specificity. The ability of serum sphingolipids to screen or diagnose HCC patients remains a question to be further explored.
High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is a powerful tool that can analyze sphingolipid metabolites with high sensitivity and specificity, and can be used in this field.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
Object of the Invention
In order to distinguish cirrhosis or hepatocellular carcinoma more effectively, the invention aims to provide application of a reagent for extracting or detecting serum Sph (d18:1) -1-P in preparing a kit for distinguishing a to-be-detected object as cirrhosis or hepatocellular carcinoma.
Solution scheme
In order to achieve the purpose of the invention, the embodiment of the invention provides the following technical scheme:
in a first aspect of the embodiments of the present invention, an application of a reagent for extracting or detecting serum Sph (d18:1) -1-P in the preparation of a kit for distinguishing a to-be-detected object as cirrhosis or hepatocellular carcinoma is provided.
In a second aspect of the embodiments of the present invention, an application of serum Sph (d18:1) -1-P in preparing or screening a reagent for distinguishing a to-be-detected object from cirrhosis or hepatocellular carcinoma is provided, where the reagent for distinguishing a to-be-detected object from cirrhosis or hepatocellular carcinoma is a reagent for extracting or detecting serum Sph (d18:1) -1-P.
In one possible implementation manner, the method for distinguishing the to-be-detected object as cirrhosis or hepatocellular carcinoma comprises the following steps: obtaining a serum sample from a to-be-detected object, and detecting the level of serum Sph (d18:1) -1-P in the serum sample by HPLC-MS/MS; the resulting serum Sph (d18:1) -1-P levels were compared to reference values.
In one possible implementation, when the cause of liver cirrhosis or hepatocellular carcinoma of the subject is unknown, the step of determining the cause of liver cirrhosis or hepatocellular carcinoma of the subject is not included before the detection of the serum Sph (d18:1) -1-P level in the serum sample;
when the etiology of liver cirrhosis or hepatocellular carcinoma of a subject to be detected before the serum Sph (d18:1) -1-P level in a serum sample is detected is known, the etiology is one or more of hepatitis B, hepatitis C, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis and hepatic vein occlusive disease; alternatively, the etiology is one or more of hepatitis c, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis, hepatic vein occlusive disease.
In one possible implementation, the test subject is negative for alpha-fetoprotein.
In one possible implementation, when the level of the obtained serum Sph (d18:1) -1-P is compared with a reference value, the reference value of the serum Sph (d18:1) -1-P is 56.29pmol/0.1ml, and when the level of the serum Sph (d18:1) -1-P is more than 56.29pmol/0.1ml, hepatocellular carcinoma is judged; when the level of serum Sph (d18:1) -1-P was less than 56.29pmol/0.1ml, it was judged to be cirrhosis.
In one possible implementation, the object to be tested is a Chinese population; optionally: the object to be tested is selected from one of the following: chinese population with unknown causes of liver cirrhosis or hepatocellular carcinoma, wherein the causes of liver cirrhosis or hepatocellular carcinoma are one or more of hepatitis B, hepatitis C, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis, and hepatic vein occlusive disease; further optionally, the object to be tested is selected from one of: the Chinese population with unknown causes of the liver cirrhosis or the hepatocellular carcinoma, wherein the causes of the liver cirrhosis or the hepatocellular carcinoma are one or more of hepatitis C, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis and hepatic vein occlusive disease.
In one possible implementation, a serum sample containing serum Sph (d18:1) -1-P was included as a positive control in a kit for distinguishing between cirrhosis and hepatocellular carcinoma.
In a third aspect of the embodiments of the present invention, there is provided a biomarker for distinguishing a subject to be detected as liver cirrhosis or hepatocellular carcinoma, the biomarker being selected from one, two, three, four, five, six, or more of the following biomarkers: cer (d18:1/26:0), Cer (d18:1/26:1), Cer (d18:1/16:1) -1-P, HexCer (d18:1/18:0), HexCer (d18:1/18:1), dhSph (d18:1/18:0), dhSph (d18:1/18:0) -1-P, dhCer (d18:0/24:1) -1-P, HexSph (d18:1), Sph (d18:1) -1-P.
In one possible implementation, the biomarker consists of Sph (d18:1) -1-P and one, two, three, four or more selected from Cer (d18:1/26:0), Cer (d18:1/26:1), Cer (d18:1/16:1) -1-P, HexCer (d18:1/18:0), HexCer (d18:1/18:1), dhSph (d18:1/18:0), dhSph (d18:1/18:0) -1-P, dhCer (d18:0/24:1) -1-P, HexSph (d18: 1).
Advantageous effects
The up-regulation of serum Sph (d18:1) -1-P in the examples of the invention shows good diagnostic performance on HCC. In particular Sph (d18:1) -1-P can also be used as a biomarker for diagnosing AFP-negative HCC. These findings are helpful in the non-invasive diagnosis of HCC, including AFP-negative HCC. In the embodiment of the invention, the serum Sph (d18:1) -1-P is used as a biomarker, and liver cancer and liver cirrhosis of all etiologies can be distinguished, so that doctors can screen HCC without confirming the etiology of people to be tested, and the application is more convenient and wider.
In the embodiment of the invention, 10 selected biomarkers which can be widely used for diagnosing hepatocellular carcinoma and can be used for AFP negative hepatocellular carcinoma are selected. In addition to Sph (d18:1) -1-P, they can also be used alone or in combination with one another for distinguishing subjects as cirrhosis or AFP-negative hepatocellular carcinoma, one, two, three, four or more than four of the remaining 9 biomarkers Cer (d18:1/26:0), Cer (d18:1/26:1), Cer (d18:1/16:1) -1-P, HexCer (d18:1/18:0), HexCoer (d18:1/18:1), dhSph (d18:1/18:0), dhSph (d18:1/18:0) -1-P, dhCer (d18:0/24:1) -1-P, HexSph (d18:1) can be combined with Sph (d18:1) -1-P to be used for distinguishing a subject to be detected as liver cirrhosis or AFP negative hepatocellular carcinoma.
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One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a diagram of the metabolic pathways of sphingolipids, which contains all 57 sphingolipids detected in the examples of the present invention.
Fig. 2A and 2B are comparative graphs of serum sphingolipid levels, wherein: serum sphingolipids with significant differences between liver cirrhosis and HCC patients are given in fig. 2A; the significant differences in serum sphingolipids between liver cirrhosis and AFP-negative HCC patients are given in figure 2B. Data are presented as mean ± SEM. (P <0.05, P <0.01, Mann-Whitney-U assay).
FIGS. 3A and 3B are schematic diagrams of biomarker identification by OPLS-DA, with the abscissa representing score values for principal components and the ordinate representing score values for orthogonal components; wherein: FIG. 3A is a graph of scores obtained from patients with cirrhosis and HCC; figure 3B is a graph of scores obtained from patients with cirrhosis and AFP-negative HCC. The different colors of the spots represent different groups that are well separated based on the levels of sphingolipids, indicating a significant difference in serum sphingolipids between these groups.
Fig. 4A and 4B are ROC curve analyses, in which: FIG. 4A is a ROC curve analysis of serum Sph (d18:1) -1-P to differentiate HCC from cirrhosis patients; FIG. 4B is a ROC curve analysis of serum Sph (d18:1) -1-P to distinguish APF-negative HCC from cirrhosis patients.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
In this study, the inventors used HPLC-MS/MS to quantify 57 serum sphingolipid metabolites from 72 HCC patients (including 24 AFP-negative HCC) and 104 liver cirrhosis patients, in order to explore the expression profile of serum sphingolipid metabolites from HCC patients and to find sphingolipids that can specifically and sensitively distinguish HCC (including AFP-negative HCC) from liver cirrhosis due to various causes, i.e., to find sphingolipid markers that can distinguish HCC from liver cirrhosis in a generally applicable sense, rather than HCC and liver cirrhosis that must be directed to specific causes.
Sphingolipid system nomenclature: sph (d18:1) or Sphingosine (d18:1) represents Sphingosine, the "d" in parentheses of the Sphingosine name represents dihydroxy, and the two numbers separated by colon thereafter represent the number of carbon atoms and the number of unsaturated bonds, respectively, of Sphingosine. dhSph (d18:0) represents sphinganine. Dihydroceramide (dhCer) and ceramide (Cer) are formed by connecting a fatty acid alkyl chain of 14 carbons to 26 carbons to the amino groups of sphinganine and sphingosine, respectively, and for the designation of the fatty acid alkyl chain in the names of dhCer and Cer, the case of the connected sphinganine or sphingosine is indicated before the slash; after the slash, the two numbers separated by the colon represent the number of carbon atoms and the number of unsaturated bonds in the fatty acid alkyl chain, respectively, e.g., Cer (d18:1/24:1) represents sphingosine attached to a fatty acid alkyl chain containing 24 carbon atoms and one unsaturated bond. Dihydroceramide (dhCer) can be desaturated with 4, 5-E-to yield ceramide (Cer). Ceramide (Cer) de-N-acylation can produce sphingosine, which can be further phosphorylated by sphingosine kinase to produce sphingosine-1-phosphate (Sph (d18:1) -1-P).
Example 1 patient population and clinicopathologic data Collection
From 7 months in 2014 to 5 months in 2015, 176 patients were successively included in the study at the center of the suspected liver disease and artificial liver in the Beijing Youyan Hospital affiliated to the university of capital medical science in China.
Patient population inclusion and exclusion criteria
(1) Diagnosis of HCC: according to Chinese guidelines for diagnosis and treatment of primary liver cancer and EASL-EORTC clinical management guidelines for hepatocellular carcinoma. Cirrhosis is diagnosed according to established criteria, mainly based on clinical data from ultrasound, CT, MRI imaging, impaired liver function and/or histological evidence. No HCC patients received local or systemic treatment of HCC (including intervention, surgery, chemoradiotherapy, targeted drug therapy) prior to blood sample collection with the aim of eliminating the effect of the treatment on sphingolipid metabolites, and some HCC patients have not been diagnosed with cirrhosis. Exclusion criteria were as follows: with other severe active psychosomatic diseases including uncontrolled primary renal, cardiac, pulmonary, vascular, neurological, metabolic diseases (hyperthyroidism, severe diabetes and adrenal disorders); abnormal lipid metabolism: severe hyperlipidemia, lipid storage disease, obesity (body Mass index BMI not less than 25 kg/m)2) Immunodeficiency disease or other systemic tumors; gestation or lactation.
Tumor size is defined based on histopathological features or medical images as the maximum diameter of a single lesion mass or the sum of two maximum tumor diameters of multiple lesions. In this study, patients were divided into two groups according to tumor size (tumor size ≦ 2cm and >2 cm). TNM staging is defined according to the united states cancer liver cancer TNM staging joint committee as follows:
according to the clinical liver cancer (BCLC) staging system of Barcelona, the diseases are divided into early stage (A), middle stage (B), late stage (C) and terminal stage (D).
Diagnostic criteria for AFP-negative HCC: HCC with serum AFP concentration less than or equal to 20 ng/ml.
(2) Diagnostic criteria for cirrhosis: imaging, biochemical or hematological examination for evidence of hepatocyte synthesis dysfunction or portal hypertension (e.g., splenic hyperactivity and esophageal fundic varices), or histological compliance with cirrhosis diagnosis. All patients were screened for HCC by imaging or liver perforation pathology.
The study according to the declaration of Helsinki. The study protocol was approved by the ethical review committee of the beijing youan hospital, affiliated with the university of capital medicine. All participants provided written consent.
Clinical features, biochemical detection and scoring system
Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) were measured using an Olympus automated biochemical analyzer AU5400(Olympus, tokyo, japan); AFP levels were determined using automated electrochemiluminescence immunoassay (Roche Diagnostics, Shanghai, China).
The study evaluated various non-invasive markers for inclusion in the population to evaluate liver fibrosis (fibrosis 4 index FIB-4, aspartate aminotransferase to platelet ratio index APRI) and liver disease severity (TBIL, ALB, end-stage liver disease model MELD, etc.) in HCC and cirrhosis patients, all calculated from clinical and laboratory parameters at the time of inclusion. The MELD scoring system is described in literature 1: kamath PS, Wiesner RH, Malinchoc M, et al.A model to predict clearance in pages with end-stage lift disease, Hepatology 2001; 33: 464-470; document 2: adnan Said, John Williams, Jermey Holden, et al model for end stage lift scale prediction access a broad spectrum of lift scale J Heastol, 2004,40(6): 897-. For APRI and FIB-4scoring systems, see literature 3: w Ray Kim, Thomas Berg, Tarik Asselah, et al.evaluation of APRI and FIB-4 sequencing systems for non-innovative assessment of pathological fibrosis in respiratory pathologies B Patents J Hepatol, 2016 Apr; 64(4) 773-780; document 4: the Chinese medical society infects the division of etiology, the division of hepatopathy of the Chinese medical society, the guide for preventing and treating chronic hepatitis B (2019 edition), the Chinese experiment and the journal of clinical infectious diseases (electronic edition), 2019,13(6):441 and 466.
Statistical analysis
Data were analyzed using the IBM SPSS statistical software package 22.0(SPSS inc., Chicago, IL, USA). P <0.05 in the two-tailed test is considered statistically significant unless otherwise stated. Continuous variables are expressed as mean ± standard deviation/standard error or median (range), depending on whether the data obeys a normal distribution. The dichotomous variables are expressed as numbers or percentages. Univariate analysis was performed based on data characteristics, and successive quantitative data between the two groups were compared using the t-test or the Mann-Whitney-U test. Comparisons between the count data sets were made using the chi-square test.
Demographic, clinical characteristics, biochemical detection, systematic scoring results for patient populations
A total of 72 HCC patients (including 24 serum AFP-negative HCC patients) and 104 cirrhosis patients were included in this analysis. In these groups, differences in gender, age, and liver function (including ALT, AST, TBIL, DBIL, TP, ALB) were not significant (all P > 0.05). The MELD score was significantly higher in patients with cirrhosis than in patients with HCC. This is readily understood by the fact that: patients with cirrhosis of the liver often seek medical attention for various complications, but HCC patients who were included in the study were diagnosed for the first time. The cirrhosis and HCC groups had 75 (72.12%) and 49 (68.06%) male patients, respectively, with mean ages of 54.26 and 56.28 years, respectively. The causes of cirrhosis and HCC patients mainly include HBV (47.12% vs 58.33%), HCV (14.42% vs 6.94%), alcoholic liver disease (16.34% vs 4.17%) and other causes (22.12% vs 30.56%). Detailed demographic and clinical characteristics of the participants are given in table 1.
Data are expressed as mean. + -. SD, median (range) or n (%) in Table 1.
Showing a comparison between cirrhosis and HCC.
A comparison between cirrhosis and AFP negative HCC is shown. P<0.05 has statistical significance. *: other causes include primary biliary cirrhosis, cryptogenic cirrhosis, hepatic vein occlusive disease, liver disease with two or more causes, and the like. Abbreviations: HCC: hepatocellular carcinoma; SD: standard deviation; AFP: alpha-fetoprotein; ALT: alanine aminotransferase; AST: an aspartate aminotransferase; TBIL: total bilirubin; DBIL: direct bilirubin; TP: total protein; ALB: albumin; MELD: end-stage liver disease model; FIB-4: fibrosis 4 score; APRI: an aspartate aminotransferase to platelet ratio index; ALD: alcoholic liver disease.
Example 2 measurement of serum sphingolipid metabolites
Detection of serum sphingolipids: determination of serum sphingolipid metabolites was performed using HPLC-MS/MS using a Spectra C8SR column (150X 3.0mm) for HPLC chromatography; 3 micron particle size, Peeke Scientific), mobile phase a is 2mM ammonium formate solution with 0.2% formic acid in water as solvent, mobile phase B is I mM ammonium formate solution with 0.2% formic acid in methanol as solvent, mobile phase elution procedure is as follows:
HPLC-MS/MS was performed using an Agilent 6410B triple quadrupole mass spectrometer (Agilent Technologies inc., Santa Clara, CA) comprising a triple quadrupole mass analyzer equipped with an electrospray ionization interface and an Agilent 1200 RRLC system (HPLC-MS/MS); the parameters of the mass spectrum are: the polarity is positive, the gas temperature is 350 ℃, the gas flow is 6L/min, the atomizer is 15psi, and the capillary is 4000V.
And (3) data analysis: data were analyzed using the IBM SPSS statistical software package 22.0(SPSS inc., Chicago, IL, USA). P <0.05 in the two-tailed test is considered statistically significant unless otherwise stated. Continuous variables are expressed as mean ± standard deviation/standard error or median (range), depending on whether the data obeys a normal distribution. The dichotomous variables are expressed as numbers or percentages. Univariate analysis was performed based on data characteristics, and successive quantitative data between the two groups were compared using the t-test or the Mann-Whitney-U test. Comparisons between the count data sets were made using the chi-square test. Multiple logistic regression analysis (forward stepwise) was performed to find sphingolipid metabolites that were independently associated with total HCC or AFP negative HCC, with P values for inclusion and removal set at 0.05 and 0.1, respectively. Correlation analysis was performed using Spearman scale test. The diagnostic performance of serum biomarkers was calculated by area under the Receiver Operating Characteristic Curve (ROC).
The orthogonal partial least squares discriminant analysis (OPLS-DA), a multivariate analysis, was used to visually distinguish HCC or AFP negatives using SIMCA 13.0 software (Umetrics, Umea, Sweden)HCC patients and cirrhosis patients. Sphingolipid data were mean centered and UV scaled. The quality of each OPLS-DA model was verified using R2Y (cum) and Q2(cum) values, which were used (between 0 and 1) to evaluate the stability and predictability of the model, respectively. The criteria for selection of biomarkers are as follows: the variable projection importance value is greater than 1; the uncertainty of the cutting method is not zero; the absolute value of P-corr in S-plot is 0.58. Cross-validation parameter Q is calculated in an orthogonal partial least squares discriminant analysis by permutation test using 100 random permutations2To test the effectiveness of the model against overfitting.
1. Serum sphingolipid profile of HCC or cirrhosis patients
There were 24 sphingolipid metabolites that differed significantly between HCC and cirrhosis patients (P <0.05), including 3 down-and 21 up-regulated sphingolipid metabolites in HCC patients. Detailed characteristics of the profile of serum sphingolipids in patients with HCC or cirrhosis are given in table 2 and fig. 2A.
Sphingolipid level units in table 2: pmol/0.1mL serum. Data are represented in median (range).
From the results of the orthogonal partial least squares discriminant analysis (OPLS-DA), Sph (d18:1) -1-P was found to distinguish HCC from cirrhosis, with R2Y (cum) and Q2(cum) being 0.717 and 0.604, respectively (FIG. 3A). The diagnostic performance of this serum sphingolipid was assessed by Roc analysis and its AUC was 0.85 (95% CI: 0.79-0.91) (P < 0.001). When the cut-off value of Sph (d18:1) -1-P was set to 56.29pmol/0.1ml, the sensitivity and specificity were 79.20% and 78.70%, respectively.
To compare the diagnostic value of Sph (d18:1) -1-P with AFP, we also evaluated the AUC of AFP, which was 0.83 (95% CI: 0.77-0.90) (P < 0.001). When the cut-off value for Sph (d18:1) -1-P was set to 20.32ng/ml, the sensitivity and specificity were 66.70% and 88.80%, respectively.
Although the AUC for Sph (d18:1) -1-P was higher than AFP, the area under the subject's working curve (AUC) was 0.85 and 0.83, respectively, but the difference was not significant (P >0.05) (FIG. 4A). Sph (d18:1) -1-P and AFP did not differ significantly in their efficacy in distinguishing HCC from cirrhosis.
2. Serum sphingolipid profile of AFP negative HCC or liver cirrhosis patients
The difference in baseline clinical parameters between AFP negative HCC and cirrhosis was not significant (P >0.05, table 1). Sixteen sphingolipid metabolites exhibited significant differences (P <0.05), including 5 down-regulated and 11 up-regulated sphingolipids. Detailed information of serum sphingolipid profiles in AFP-negative HCC patients is shown in table 2 and figure 2B.
OPLS-DA was performed to distinguish AFP negative HCC from cirrhosis. The results show that Sph (d18:1) -1-P can also distinguish AFP negative HCC from cirrhosis patients, with R2Y (cum) and Q2(cum) being 0.568 and 0.464, respectively (fig. 3B). The AUC for Sph (d18:1) -1-P was 0.79 (95% CI: 0.68-0.90) (P <0.001), and when the cut-off for Sph (d18:1) -1-P was set to 56.29pmol/0.1ml, the sensitivity and specificity were 62.50% and 77.90%, respectively (FIG. 4B).
3. Correlation of serum Sph (d18:1) -1-P with clinical parameters
AFP levels were significantly lower in patients with cirrhosis than in HCC patients (P <0.01), while the difference in AFP levels between cirrhosis and AFP-negative HCC patients was not significant (P > 0.05). Spearman scale correlation analysis showed that the level of serum Sph (d18:1) -1-P, respectively, was not correlated with AFP in liver cirrhosis (r ═ 0.04, P ═ 0.71), total HCC (r ═ 0.06, P ═ 0.60), AFP positive HCC (r ═ 0.09, P ═ 0.56) or AFP negative HCC (r ═ 0.13, P ═ 0.55).
To investigate the relationship between Sph (d18:1) -1-P and tumor size, we divided HCC patients into two groups according to tumor size (tumor size ≦ 2cm and >2 cm). Serum Sph (d18:1) -1-P levels were not statistically significantly different between the two groups (58.09 ± 3.09vs 58.11 ± 2.54pmol/0.1ml, P ═ 0.476). Furthermore, we divided HCC patients into four groups, TNM I, TNM II, TNM III and TNM IV, according to the TNM staging system. Serum levels of Sph (d18:1) -1-P were 58.18. + -. 2.91, 57.78. + -. 1.57, 58.74. + -. 3.15 and 58.20. + -. 3.31pmol/0.1ml, respectively. There was no difference between these groups (P ═ 0.771). Furthermore, the difference between the two groups was also not significant (all P > 0.05). HCC patients were then divided into four groups, A, B, C and D, according to the BCLC staging system. Serum Sph (d18:1) -1-P levels were 58.45. + -. 2.81, 57.91. + -. 1.62, 59.10. + -. 3.50 and 58.30. + -. 3.15pmol/0.1ml, respectively. Also, the difference between the two groups was not significant (all P > 0.05).
Studies have found that patients with AFP-negative HCC are less likely to develop cirrhosis nodules and a poorer Edmondson-Steiner grade than patients with AFP-positive HCC, and instead they are more likely to form an intact tumor envelope and have a favorable long-term prognosis. Furthermore, we counted the number of HCC patients with distant metastases, intrahepatic metastases and portal vein infringement. The results of the analysis indicated that patients with AFP-negative HCC had lower rates of distant metastasis (P <0.001), intrahepatic metastasis (P ═ 0.007), and portal vein invasion (P ═ 0.016) than patients with AFP-positive HCC. Therefore, in order to improve the survival and prognosis of AFP negative HCC patients, it is important to identify these patients and provide treatment in a timely manner.
In the present invention, the inventors have creatively found that serum Sph (d18:1) -1-P can distinguish HCC (including AFP-negative HCC) from cirrhosis. To our knowledge, this is the first report on the ability of Sph (d18:1) -1-P to effectively distinguish AFP-negative HCC from cirrhosis.
In this study, we found that 10 sphingolipids including Sph (d18:1) -1-P showed significant differences in both AFP-negative HCC and cirrhosis, and HCC and cirrhosis. In addition to 2 hexchers, the remaining 8 sphingolipids in AFP-negative HCC patients are upregulated and most of them belong to long-chain or ultralong-chain sphingolipids. This finding lays a preliminary foundation for further study of the pathogenesis of HCC and AFP-negative HCC.
Notably, we detected more types and amounts of sphingolipids, which provides more opportunities and convincing evidence for finding the best diagnostic biomarkers. In addition, we found that the up-regulation of Sph (d18:1) -1-P is most closely related to HCC in chinese population, and the diversity of different etiologies, regions, ethnicities, etc. can influence the selection of biomarkers.
Interestingly and importantly, our findings first demonstrated that Sph (d18:1) -1-P can effectively distinguish AFP-negative HCC from cirrhosis in liver. Furthermore, we found that levels of Sph (d18:1) -1-P in patients with liver cirrhosis or HCC were independent of AFP concentration, and serum Sph (d18:1) -1-P was not associated with AFP in patients with liver cirrhosis, AFP-positive HCC, and AFP-negative HCC. This suggests that the regulatory mechanism of Sph (d18:1) -1-P may be independent of AFP in HCC patients. Our results also show that the difference in serum Sph (d18:1) -1-P levels between the two groups was not statistically significant (tumor size ≦ 2cm or >2cm, P ═ 0.476). Also, there was no difference between patients with different TNM stages and BCLC stages. Thus, from these results, it can be concluded that Sph (d18:1) -1-P can be used to identify various types of HCC patients, but not tumor size, TNM stage, and BCLC stage.
In conclusion, up-regulation of serum Sph (d18:1) -1-P shows good diagnostic performance against HCC. In particular Sph (d18:1) -1-P can also be used as a biomarker for diagnosing AFP-negative HCC. These findings are helpful in the non-invasive diagnosis of HCC, including AFP-negative HCC.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. Application of a reagent for extracting or detecting serum Sph (d18:1) -1-P in preparation of a kit for distinguishing a to-be-detected object from cirrhosis or hepatocellular carcinoma.
2. Application of serum Sph (d18:1) -1-P in preparation or screening of a reagent for distinguishing a to-be-detected object from cirrhosis or hepatocellular carcinoma, wherein the reagent for distinguishing the to-be-detected object from cirrhosis or hepatocellular carcinoma is a reagent for extracting or detecting the serum Sph (d18:1) -1-P.
3. The use according to claim 1 or claim 2, wherein: the method for distinguishing the liver cirrhosis or hepatocellular carcinoma of the test object comprises the following steps: obtaining a serum sample from a to-be-detected object, and detecting the level of serum Sph (d18:1) -1-P in the serum sample by HPLC-MS/MS; the resulting serum Sph (d18:1) -1-P levels were compared to reference values.
4. Use according to claim 3, characterized in that:
when the cause of the liver cirrhosis or hepatocellular carcinoma of the subject is unknown, the step of determining the cause of the liver cirrhosis or hepatocellular carcinoma of the subject is not included before the detection of the serum Sph (d18:1) -1-P level in the serum sample;
when the etiology of liver cirrhosis or hepatocellular carcinoma of a subject to be detected before the serum Sph (d18:1) -1-P level in a serum sample is detected is known, the etiology is one or more of hepatitis B, hepatitis C, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis and hepatic vein occlusive disease; alternatively, the etiology is one or more of hepatitis c, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis, hepatic vein occlusive disease.
5. Use according to claim 1 or 2, characterized in that: the alpha fetoprotein of the object to be detected is negative.
6. Use according to claim 1, characterized in that: comparing the obtained serum Sph (d18:1) -1-P level with a reference value, wherein the reference value of the serum Sph (d18:1) -1-P is 56.29pmol/0.1ml, and when the serum Sph (d18:1) -1-P level is more than 56.29pmol/0.1ml, the hepatocellular carcinoma is judged; when the level of serum Sph (d18:1) -1-P was less than 56.29pmol/0.1ml, it was judged to be cirrhosis.
7. Use according to claim 1 or 2, characterized in that: the object to be detected is Chinese population; optionally: the object to be tested is selected from one of the following: chinese population with unknown causes of liver cirrhosis or hepatocellular carcinoma, wherein the causes of liver cirrhosis or hepatocellular carcinoma are one or more of hepatitis B, hepatitis C, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis, and hepatic vein occlusive disease; further optionally, the liver cirrhosis or hepatocellular carcinoma is not known, and the liver cirrhosis or hepatocellular carcinoma is one or more of hepatitis c, alcoholic liver disease, primary biliary cirrhosis, cryptogenic cirrhosis, and hepatic vein occlusive disease.
8. Use according to claim 1, characterized in that: a kit for differentiating between cirrhosis and hepatocellular carcinoma included a serum sample containing serum Sph (d18:1) -1-P as a positive control.
9. A biomarker for distinguishing a subject to be tested as liver cirrhosis or AFP-negative hepatocellular carcinoma, characterized by: consisting of one, two, three, four, five, six or more biomarkers selected from the following: cer (d18:1/26:0), Cer (d18:1/26:1), Cer (d18:1/16:1) -1-P, HexCer (d18:1/18:0), HexCer (d18:1/18:1), dhSph (d18:1/18:0), dhSph (d18:1/18:0) -1-P, dhCer (d18:0/24:1) -1-P, HexSph (d18:1), Sph (d18:1) -1-P.
10. The biomarker of claim 9, characterized in that: consists of Sph (d18:1) -1-P and one, two, three, four or more selected from Cer (d18:1/26:0), Cer (d18:1/26:1), Cer (d18:1/16:1) -1-P, HexCer (d18:1/18:0), HexCor (d18:1/18:1), dhSph (d18:1/18:0), dhSph (d18:1/18:0) -1-P, dhCer (d18:0/24:1) -1-P, HexSph (d18: 1).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113295793A (en) * | 2021-05-20 | 2021-08-24 | 复旦大学附属中山医院 | Biomarker for predicting early diabetes and diabetes occurrence, detection method and application thereof |
| WO2023283410A3 (en) * | 2021-07-09 | 2023-03-23 | Board Of Regents, The University Of Texas System | Exosomal lipids and metabolites for the early detection of hepatocellular carcinoma |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484527A (en) * | 2012-06-08 | 2014-01-01 | 上海市肿瘤研究所 | Uses of serum DKK1 in preparation of hepatocellular carcinoma and alpha-fetoprotein positive chronic liver disease differential diagnosis regents |
| CN106383231A (en) * | 2016-09-02 | 2017-02-08 | 四川大学 | Marker combination used for liver cancer diagnosis, and application thereof |
| CN107454940A (en) * | 2015-04-13 | 2017-12-08 | 法兰克福大学 | The serum biomarkers of hepatocellular carcinoma (HCC) |
-
2020
- 2020-08-25 CN CN202010865122.2A patent/CN112034171A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484527A (en) * | 2012-06-08 | 2014-01-01 | 上海市肿瘤研究所 | Uses of serum DKK1 in preparation of hepatocellular carcinoma and alpha-fetoprotein positive chronic liver disease differential diagnosis regents |
| CN107454940A (en) * | 2015-04-13 | 2017-12-08 | 法兰克福大学 | The serum biomarkers of hepatocellular carcinoma (HCC) |
| CN106383231A (en) * | 2016-09-02 | 2017-02-08 | 四川大学 | Marker combination used for liver cancer diagnosis, and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BAASANJAV URANBILEG等: "Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence", PLOS ONE, pages 1 - 16 * |
| 蒋莹莹等: "血清鞘脂用于乙肝相关甲胎蛋白阴性肝细胞癌的诊断价值研究", 胃肠病学和肝病学杂志, vol. 27, no. 5, pages 563 - 568 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113295793A (en) * | 2021-05-20 | 2021-08-24 | 复旦大学附属中山医院 | Biomarker for predicting early diabetes and diabetes occurrence, detection method and application thereof |
| WO2023283410A3 (en) * | 2021-07-09 | 2023-03-23 | Board Of Regents, The University Of Texas System | Exosomal lipids and metabolites for the early detection of hepatocellular carcinoma |
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