MARKERS FOR DIAGNOSIS AND PROGNOSTIC PREDICTION OF NPC AND APPLICATION THEREOF

Abstract

Disclosed are markers for nasopharyngeal carcinoma (NPC) diagnosis and prognostic prediction, and use thereof. The INSL5 level in a plasma sample can be determined by means of ELISA. It was found that the INSL5 plasma level is significantly different between NPC patients and healthy population. The analysis results of a ROC curve show that an area under curve of INSL5 is 0.941, the critical value of the INSL5 level is 2.45 ng/ml, and the sensitivity and specificity thereof are 93.2% and 81.5% respectively. In the healthy cohort, the INSL5 level in EBV-positive plasma is significantly higher than that in EBV-negative plasma. The EBV-negative subjects comprise 34 healthy individuals and 72 NPC patients, and the INSL5 levels are observed to be significantly different between these two cohorts. The results of the ROC curve show that the area under curve of INSL5 is 0.988, the critical value of the INSL5 level is 2.25 ng/ml, and the sensitivity and specificity thereof are 97.2% and 91.2% respectively. Thus, the present disclosure can efficiently distinguish EBV-negative normal people from NPC patients.

Claims

1. A method for nasopharyngeal carcinoma (NPC) diagnosis and prognostic prediction, said method comprising: using Insulin-like factor 5 (INSL5) as a marker.

2. A method for nasopharyngeal carcinoma (NPC) diagnosis and prognostic prediction, said method comprising: using a reagent for quantifying a plasma concentration of Insulin-like factor 5 (INSL5) in the preparation of an agent.

3. The method of claim 2, wherein the reagent for quantifying the plasma concentration of Insulin-like factor 5 (INSL5) is selected from INSL5-specific ELISA detection reagents.

4. The method of claim 2, wherein a criterion for high risk of nasopharyngeal carcinoma (NPC) is determined by plotting a ROC curve, and a criterion for poor prognosis of nasopharyngeal carcinoma is determined by a Kaplan-Meier survival analysis.

5. The method of claim 4, wherein the criterion for poor prognosis of NPC is determined by the Kaplan-Meier survival analysis with combining INSL5 with an EBV DNA copy number in plasma.

6. A method for nasopharyngeal carcinoma (NPC) diagnosis and prognostic prediction, said method comprising: determining an INSL5 concentration in a plasma sample to be tested; determining whether the plasma sample to be tested is a high-risk sample of NPC; and predicting prognosis thereof based on the determined INSL5 concentration.

7. The method of claim 6, further comprising: determining a criterion for high risk of NPC by plotting a ROC curve, and determining a criterion for poor prognosis of NPC through either a Kaplan-Meier survival analysis, or through the Kaplan-Meier survival analysis with combining INSL5 with a EBV DNA copy number in plasma.

8. The method of claim 7, wherein the criterion for high risk of NPC is a Yuden index of the ROC curve.

9. The method of claim 7, wherein, determining to have high risk of NPC when the INSL5 concentration is higher than 2.45 ng/ml; determining to have poor prognosis of NPC when the INSL5 concentration is higher than 3.73 ng/ml, or when the INSL5 concentration is higher than 3.73 ng/ml and a EBV DNA copy number is higher than 4000 copies/ml.

10. The method of claim 6, wherein the step of determining the INSL5 concentration comprises: diluting the plasma sample in a ratio of 1:10 with a sample diluent, adding the plasma sample in a microplate 100 l/well, and incubating for 2 hours at 37 C.; washing the plate with PBST for three times, then removing PBST and adding a diluted biotin-labeled detection antibody 23G9 100 l/well, and incubating for 2 hours at 37 C.; washing the plate with PBST for three times, then removing PBST and adding a diluted streptavidin conjugated with HRP 100 l/well, and incubating for 20 minutes in dark at room temperature; washing the plate with PBST for five times, then removing PBST and adding a chromogenic substrate TMB 100 l/well, and developing for no more than 20 minutes at 37 C.; terminating the reaction by adding 50 l of 2M H.sub.2SO.sub.4 and standing for 10 min; and reading an Optical density (OD) value at wavelength of 450 nm on a microplate reader, and calculating the INSL5 concentration in the sample to be tested according to a standard curve.

11. A method for nasopharyngeal carcinoma (NPC) diagnosis and prognostic prediction, said method comprising: determining an INSL5 concentration in a plasma sample to be tested; and determining whether the plasma sample to be tested is a high-risk sample of NPC or predicting prognosis thereof based on the determined INSL5 concentration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows diagrams confirming that INSL5 antibodies 46B8 and 23G9 can bind INSL5 and have different epitopes, and thus can be used for sandwich ELISA to detect INSL5.

[0039] FIG. 2 is a scatter plot showing the concentrations of INSL5 in NPC patients, healthy control cohort with EBV-positive (Normal EBV(+)) and healthy control cohort with EBV-negative (Normal EBV()).

[0040] FIG. 3 shows the results of ROC curve analysis of the concentrations of INSL5 in NPC patients and healthy control cohort (normal).

[0041] FIG. 4 is a ROC curve which shows the diagnostic efficacy of INSL5 for EBV-negative NPC patients.

[0042] FIG. 5 is a curve which shows the results of survival prognosis analysis by means of INSL5 alone;

[0043] FIG. 6 is a curve which shows the results of survival prognosis analysis by means of the combination of INSL5 and the EBV DNA copy number.

DETAILED DESCRIPTION

[0044] Preparation of INSL5 Antibodies.

[0045] BALB/c female mice of 6-8 weeks old were routinely immunized by intraperitoneal injection of a recombinantly expressed mature INSL5 fusion protein having a human Fc tag, in 50 g/mouse/time, following by strengthening immunization every two weeks. After three times of immunization, the antibody titers in the mouse serum was determined by ELISA. Then, the mice having higher antibody titers were sacrificed after strengthening immunization once. The spleens were taken out for making cell suspension, which then fused with SP2/0 mouse myeloma cells. The antigen, which was used for the detection of antibody titers of mouse serum by ELISA, was a biotinylated mature INSL5-His fusion protein containing a 6His tag. After cell fusion, a lot of ELISA screening and subcloning were performed, and finally obtained two clones, 46B8 and 23G9, which showed stronger specific binding activity to the mature INSL5-His fusion protein. In addition, the two clones, 46B8 and 23G9, could bind to Pre-INSL5, in which the linking peptide is not completely removed. It shows that the binding epitopes of the two clones locate on A or B chain of INSL5 protein.

[0046] Buffers and Reagents for the INSL5 ELISA Test Kit.

[0047] ELISA plate: a microplate pre-coated with the antibody 46B8 for capturing INSL5. The preparation method thereof was as follows:

[0048] 1) diluting INSL5 antibody 46B8 with a coating buffer to 2 g/ml, and adding 100 l of the diluted 46B8 antibody into each well of the microplate, and incubating at room temperature overnight;

[0049] 2) spin-drying the coating buffer, washing 3 times with PBST, then removing PBST and adding a blocking solution containing 3% BSA, 300 l/well, and blocking at room temperature for 2 hours;

[0050] 3) washing the plate with PBST for 3 times, patting and removing PBST, and storing at 4 C. until use.

[0051] Coating buffer: PBS buffer, pH 7.3;

[0052] Blocking solution: 3% BSA, 1PBS;

[0053] Diluent: 1PBS, pH 7.2-7.4;

[0054] Washing solution: 1PBS, 0.05% Tween-20, pH 7.2-7.4;

[0055] Standard samples: INSL5 powder was dissolved and diluted to obtain a gradient concentration of solutions, as the standard samples, which can be purchased from Phoenix Biotech;

[0056] Detection antibody: 23G9 which was labeled with biotin purchased from Thermofisher;

[0057] Detection reagent: HRP-conjugated streptavidin, purchased from R&D

[0058] Corporation;

[0059] Chromogenic substrate: TMB, purchased from Sigma;

[0060] Elimination solution: 2M H2SO4.

[0061] Patients with Nasopharyngeal Carcinoma.

[0062] 339 cases of inpatients and outpatient with NPC were collected from Sun Yat-sen University Cancer Center, from January 2009 to December 2012. Inclusion criteria was that the patients had been confirmed to have type II/III nasopharyngeal carcinoma by pathological diagnosis (based on World Health Organization Classification System 1978) and had precise clinical stages (based on USA AJCC cancer staging manual, 7th edition); all the samples were derived from the patients from the areas having high incidence of nasopharyngeal carcinoma in southern China (e.g., Guangdong, Guangxi, Jiangxi, Hunan, Fujian, and Sichuan provinces); the plasma samples were taken before treatment; 284 of the 339 cases had complete demographic data and medical records, including the results of serum VCA-IgA titer and quantitative detection of plasma EBV DNA copies. Among the patients, there were 75 EBV-negative cases and 209 EBV-positive cases.

[0063] Healthy Population.

[0064] 65 plasma samples were randomly selected from the physical examination of staffs of Sun Yat-Sen University. The data, including the results of VCA-IgA titer, of all the healthy population were kept, with the exclusion of other diseases. Among them, there were 34 EBV-negative cases and 31 EBV-positive cases.

[0065] Detection Process.

[0066] The detection process comprised:

[0067] 1) diluting the plasma samples in the ratio of 1:10 with a sample diluent, adding the samples 100 l/well, and incubating for 2 hours at 37 C.;

[0068] 2) washing the plate with PBST for three times, then removing PBST by flicking the plate over a sink and patting the plate on a paper towel and adding the diluted biotin-labeled detection antibody 23G9 100 l/well, and incubating for 2 hours at 37 C.;

[0069] 3) washing the plate with PBST for three times, then removing PBST by flicking the plate over a sink and patting the plate on a paper towel and adding the diluted streptavidin conjugated with HRP 100 l/well, and incubating for 20 minutes in dark at room temperature;

[0070] 4) washing the plate with PBST for five times, then removing PBST by flicking the plate over a sink and patting the plate on a paper towel and adding the chromogenic substrate TMB100 l/well, and developing at 37 C. for no more than 20 minutes;

[0071] 5) terminating the reaction by adding 50 l of 2M H2SO4 and standing for 10 min;

[0072] 6) reading OD values at wavelength of 450 nm on the microplate reader, and calculating the concentrations of INSL5 in the samples to be tested according to the standard curve.

[0073] Detection Results and Analysis.

[0074] 1. Antibody Verification

[0075] The antibodies 46B8 and 23G9 against INSL5 were demonstrated to have different epitopes by competitive ELISA, thereby being capable of assembling an ELISA Kit for the detection of INSL5.

[0076] Mouse antibodies 46B8-mlgG2a and 23G9-mlgG2a, as well as human-mouse chimeric antibodies 46B8-hIgG1 and 23G9-hIgG1 were recombinantly expressed by the inventors. As shown in FIGS. 1A and 1B, the four recombinantly expressed antibodies had good binding ability to the antigen Pre-INSL5 protein. Then, it was found that 46B8-hIgG1 could not compete for the binding of 23G9-mlgG2a to Pre-INSL5 antigen (FIG. 1C), and 23G9-hIgG1 also could not compete for the binding of 46B8-mlgG2a to Pre-INSL5 antigen (FIG. 1D). Further, it could detect different INSL5 of different concentrations by using 46B8 as a capture antibody and 23G9 as a detection antibody (FIG. 1E).

[0077] 2. Detection of Plasma Samples.

[0078] 339 cases of NPC patients and 65 cases of healthy controls were selected for the detection of plasma samples which were derived from Sun Yat-sen University Cancer Center. The concentrations of INSL5 were calculated according to the concentrations of the standard samples, and analyzed by SPSS (20.0) statistical software. Statistical analysis showed that the data was non-normal distribution, and thus was analyzed by the Wilcoxon rank sum test.

[0079] The results of the Wilcoxon rank sum test showed that there was a statistically significant difference in the plasma concentrations of INSL5 between the two cohorts of population. The INSL5 plasma concentrations of NPC patients were significantly higher than those of the normal cohort (p value is less than 0.0001). Meanwhile, the INSL5 concentrations of the EBV-positive cohort were significantly higher than those of the EBV-negative cohort in the normal cohort, and the scatter diagram thereof was shown in FIG. 2.

TABLE-US-00001 TABLE 1 Comparison of INSL5 levels between NPC patients and normal cohorts Median concentration Interquartile P Cohorts (ng/ml) range value <0.001 Nasopharyngeal 4.1464 1.886 carcinoma Normal EBV (+) cohort 2.2854 0.7343 Normal EBV () cohort 1.6524 0.3292

[0080] A ROC curve was plotted based on the INSL5 concentrations of the NPC patients and the normal cohort (FIG. 3).

[0081] The Cut-off value was set based on the concentration of INSL5 when Yoden index reached the maximum value. In the way, the concentration of INSL5 of 2.45 ng/ml was selected as the Cut-off value based on the analytical curve. The detection sensitivity was 93.2%, the detection specificity was 81.5%, and the area under curve was 0.941, when the concentration of INSL5 was used for the NPC diagnosis.

[0082] In addition, INSL5 can clearly distinguish the NPC patients from normal people, in the EBV-negative population. A ROC curve was plotted based on the INSL5 concentrations in the EBV-negative cohort (FIG. 4). The cut-off value was set be the concentration of INSL5 when the Yoden index reached the maximum value. In this way, the concentration of INSL5 of 2.25 ng/ml was selected as the Cut-off value based on the analytical curve. The detection sensitivity was 97.2%, the detection specificity was 91.2%, and the area under curve was 0.988, when the INSL5 concentration was used for the NPC diagnosis.

[0083] With the analysis of 304 cases having prognostic information, it was found that INSL5 was an independent prognostic factor, with a critical value of 3.73 ng/ml. A higher concentration of INSL5 can predict poor prognosis. The five-year survival rate and the Disease-free survival rate were 81.3% and 88.2% respectively, which were much lower than those in the cohort with lower INSL5 concentration which had the five-year survival rate of 92.2% and Disease-free survival rate of 97.3% (FIG. 5).

[0084] In addition, the prognosis of NPC patients could be better predicted by the combination of the concentration of INSL5, and the EBV DNA copy number which has a critical value of 4000 copies/ml (FIG. 6).

[0085] In conclusion, the experimental data shows that quantitative analysis of the INSL5 plasma concentration can provide a new detection marker which has high sensitivity and specificity for the NPC diagnosis, especially can be valuable for the diagnosis of EBV-negative patients and reduce the possibility of missed diagnosis. Meanwhile, INSL5 can be used as a good independent marker for prognostic prediction, and INSL5 used in combination with the EBV DNA copy number can improve the ability of prognostic prediction. Thus, the present disclosure provides a new approach for the NPC diagnosis and prognostic prediction.