PANCREATIC CANCER DIAGNOSTIC COMPOSITION TO BE USED IN BUFFY COAT SAMPLE

Abstract

The present invention relates to: a pancreatic cancer diagnostic biomarker comprising one or more selected from the group consisting of Interleukin-28 (IL-28), Interleukin-29 (IL-29) and genes encoding same; a composition for diagnosing pancreatic cancer, comprising a preparation capable of detecting the biomarker; and a pancreatic cancer diagnostic method using the composition, and the present invention is to be used in a blood-derived buffy coat sample.

Claims

1. A composition for diagnosis of pancreatic cancer, wherein the composition comprises an agent capable of detecting at least one selected from the group consisting of interleukin 28, interleukin 29, and genes coding therefor, and is applied to a blood-derived buffy coat.

2. The composition of claim 1, wherein the buffy coat is a whole white blood cell layer formed between the upper plasma layer and the lower red blood cell layer following centrifugation and is obtained by forming an upper plasma layer, a medium buffy coat layer, and a lower red blood cell layer upon centrifugation under at least one selected from the following conditions (1) to (3) and isolating the medium buffy coat layer: (1) temperature: 2 to 30° C.; (2) speed: 300 g to 2000 g; and (3) duration time: 5 to 20 minutes.

3. The composition of claim 1, wherein the buffy coat is obtained by forming an upper plasma layer, a medium buffy coat layer, and a lower red blood cell layer upon centrifugation under at least one selected from the following conditions (1) to (3) and isolating the medium buffy coat layer: (1) temperature: 4 to 25° C.; (2) speed: 500 g to 1800 g; and (3) duration time: 7 to 20 minutes.

4. The composition of claim 1, wherein the detecting agent is at least one selected from consisting of low-molecular weight compounds, proteins, peptides, and nucleic acids, which are all able to bind to at least one selected from the group consisting of interleukin 28, interleukin 29, and genes coding therefor.

5. A kit for diagnosis of pancreatic cancer, comprising the composition of claim 1.

6. The kit of claim 5, wherein the kit is applied to a blood-derived buffy coat.

7. A method for providing information for diagnosis of pancreatic cancer, the method comprising a step of detecting at least one selected from the group consisting of interleukin 28, interleukin 29, and genes coding therefor in a blood sample isolated from a subject, wherein the blood sample is a blood-derived buffy coat.

8. The method of claim 7, further comprising, after the detecting step, a step of determining the subject to be a pancreatic cancer patient when the biomarker is present in the blood sample or when the level of the biomarker is higher in the sample than a normal reference sample.

9. The method of claim 7, wherein the buffy coat is a whole white blood cell layer formed between the upper plasma layer and the lower red blood cell layer following centrifugation and is obtained by forming an upper plasma layer, a medium buffy coat layer, and a lower red blood cell layer upon centrifugation under at least one selected from the following conditions (1) to (3) and isolating the medium buffy coat layer: (1) temperature: 2 to 30° C.; (2) speed: 300 g to 2000 g; and (3) duration time: 5 to 20 minutes.

10. The method of claim 7, wherein the buffy coat is obtained by forming an upper plasma layer, a medium buffy coat layer, and a lower red blood cell layer upon centrifugation under at least one selected from the following conditions (1) to (3) and isolating the medium buffy coat layer: (1) temperature: 4 to 25° C.; (2) speed: 500 g to 1800 g; and (3) duration time: 7 to 20 minutes.

Description

DESCRIPTION OF DRAWINGS

[0068] FIG. 1 is a graph showing expression level differences of IL-29 in buffy coat and PBMC samples obtained from pancreatic cancer patients and normal persons.

[0069] FIG. 2 is a graph showing expression level differences of IL-28 in buffy coat and PBMC samples obtained from pancreatic cancer patients and normal persons.

[0070] FIG. 3 is a schematic view illustrating a process of preparing a buffy coat sample in comparison with a PBMC sample.

[0071] FIG. 4 shows graphs of expression levels of IL-28 (upper) and IL-29 (lower) in buffy coat and PBMC samples obtained from the same pancreatic cancer patients.

[0072] FIG. 5a is a graph showing expression levels of IL-28 in buffy coat and PBMC samples obtained from pancreatic cancer patients in comparison with that that in PBMC samples from normal persons.

[0073] FIG. 5b is a graph showing expression levels of IL-28 in buffy coat and PBMC samples obtained from pancreatic cancer patients in comparison with that that in buffy coat samples from normal persons.

[0074] FIG. 6a is a graph showing expression levels of IL-29 in buffy coat and PBMC samples obtained from pancreatic cancer patients in comparison with that that in PBMC samples from normal persons.

[0075] FIG. 6b is a graph showing expression levels of IL-29 in buffy coat and PBMC samples obtained from pancreatic cancer patients in comparison with that that in buffy coat samples from normal persons.

[0076] FIG. 7a is a graph showing expression levels of IL-28 in buffy coat and PBMC samples obtained from pancreatic cancer patients in comparison with that that in samples (average value of IL-28 expression levels in PBMC and buffy coat samples) from normal persons.

[0077] FIG. 7b is a graph showing expression levels of IL-29 in buffy coat and PBMC samples obtained from pancreatic cancer patients in comparison with that that in samples (average value of IL-29 expression levels in PBMC and buffy coat samples) from normal persons.

MODE FOR INVENTION

[0078] A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.

Example 1: Sample Preparation

[0079] 1.1. Buffy Coat Sample Preparation

[0080] An examination was made of expression changes of pancreatic cancer biomarkers between pancreatic cancer samples and normal samples. In this regard, mRNA expression of IL-29 and IL-28 was analyzed in buffy coats separated from blood of pancreatic cancer patients. For comparison, the same experiment was conducted with the blood-derived buffy coats from healthy persons (hereinafter referred to as normal subjects).

[0081] First, 5 cc of blood from each of pancreatic cancer patients and normal subjects (normal control) was sampled into an EDTA tube and stored at room temperature. The blood-containing EDTA tube was centrifuged at 800 g and 4° C. for 10 min. In the tube, a plasma, a buffy coat, and red blood cells were layered in the order from the top. After removal of the plasma, the buffy coat was recovered in an amount of 250 μl and stored at −80° C.

[0082] Total RNA was extracted from 250 μl of the deep-frozen buffy coat using NucleoSpin® RNA Blood kit (MACHEREY-NAGEL) according to the recommended protocol. From 1 μg of the RNA, cDNA was synthesized. For cDNA synthesis, GoScript™ Reverse Transcription system kit (Promega) was used.

[0083] 1.2. Peripheral Blood Mononuclear Cell (PBMC) Sample Preparation

[0084] An examination was made of expression changes of pancreatic cancer biomarkers between pancreatic cancer samples and normal samples. In this regard, mRNA expression of IL-29 and IL-28 (IL-28A) was analyzed in peripheral blood mononuclear cells from blood of pancreatic cancer patients. For comparison, the same experiment was conducted with the peripheral blood mononuclear cells from healthy persons (hereinafter referred to as normal subjects).

[0085] First, 5 cc of blood from each of pancreatic cancer patients and normal subjects (normal control) was sampled into an EDTA tube and stored at room temperature. When blood is high in viscosity, it is difficult to separate mononuclear cells from the blood. Thus, the blood sample was diluted at a ratio of 1:1 with 1×PBS. The lymphocyte separation medium (Lymphosep™, L0560-500, Biowest) was prepared as a density gradient medium in a 15-ml conical tube, followed by adding the blood sample on Ficoll, with care to prevent mingling with the separation medium. Centrifugation at 800 g for 30 min with minimum acceleration and deceleration resulted in separating the blood into plasma, peripheral blood mononuclear cells (PBMC), Lymphosep, granulocytes, and erythrocytes in the density-increasing order from the top to the bottom in the tube. The medium white cell layer (monocytes) was recovered. The cells were added with PBS, centrifuged at 400 g for 3 min, and washed twice before use. The residual cells were stored at −80° C.

[0086] The PBMC thus prepared was added with 1 ml of Trizol to suspend the adherent cells. After 200 μl of chloroform, centrifugation was conducted at 12,000 g for 15 min. Only the transparent uppermost supernatant was picked out and mixed with an equal volume of isopropanol before centrifugation at 12,000 g for 15 min. After the supernatant was discarded, the pellet was washed with 1 ml of 75% ethanol by centrifugation at 12,000 g for 10 min. The supernatant was discarded and the pellet was sufficiently dried for 1 hour. The RNA pellet was added with 30 μl of nuclease-free water before RNA to quantitation. For cDNA synthesis, 1 μg of RNA was used. cDNA synthesis was carried out using GoScript™ Reverse Transcription system kit (Promega).

Example 2. Real-time qPCR

[0087] Gene expression in the samples prepared in Example 1 was measured by real-time qPCR. The real-time qPCR was carried out in a probe-based multiplex PCR assay. For multiplex PCR, respective probes for the markers IL-29 (GenBank Accession No. NM_172140.2) and IL-28A (GenBank Accession No. NM_172138.2; hereinafter referred to as “IL-28”) were labeled with FAM dye while GAPDH for use as an internal reference gene was labeled with HEX dye. Primers and probes were purchased from IDT (Integrated DNA Technologies, Inc.).

[0088] To identify the expression of each gene in the samples, a reaction mix including GoTaq® Probe qPCR Master Mix (Promega) was prepared in a final volume of 20 μl according to the protocol. Gene expression assay was performed using QuantStudio 3 and 5 Real-Time PCR system instrument (Applied Biosystems) under the standard cycling condition provided by the software of the instrument. Real-time qPCR results were expressed as ΔCt values, which were differences between Ct values, that is, mRNA expression levels of the markers IL-29 and IL-28 and the internal reference gene GAPDH. All data for comparison of mRNA expression were analyzed in terms of ΔCt.

[0089] Nucleotide sequences (5′.fwdarw.3′) of the primers and probes used are summarized in Table 2, below.

TABLE-US-00002 TABLE 2 SEQ ID Sequence (5′.fwdarw.3′) NO: IL-29_F GGT TCA AAT CTC TGT CAC CAC A  4 primer IL-29_R GAA GAC AGG AGA GCT GCA AC  5 primer IL-28A_F CAG CCT CAG AGT GTT TCT TCT  6 primer IL-28A_R TCC AGT CAC GGT CAG CA  7 primer GPC-1_F GTC ATG AAG CTG GTC TAC TG  8 primer GPC-1_R AGC CCT TGA GCA CAT TTC  9 primer IL-29_Probe FAM/TCAAGAAGG/ZEN/CCAGGGACGCC/IBFQ 10 IL-28_Probe FAM- 11 TCATGTCTA/ZEN/GTTTCATTCCTGATCTCTGGTCT- IBFQ

[0090] (in Table 2, the probes were structured to include 5′ FAM dye, internal ZEN Quencher, and 3′ Iowa Black® Fluorescent Quencher (IBFQ))

Example 3: Measurement of Marker Expression in Each Sample

[0091] Results of the real-time qPCR performed in Example 2 are depicted in FIGS. 1 and 2.

[0092] FIG. 1 is a graph showing expression level differences of IL-29 in buffy coat samples obtained from 52 pancreatic cancer patients and 44 normal persons and in PBMC samples obtained from 54 pancreatic cancer patients and 19 normal persons. As can be seen in FIG. 1, the average ΔCt value of IL-29 was significantly reduced in the buffy coat samples from the pancreatic cancer patients, compared to the normal persons. In contrast, the average ΔCt value of IL-29 in the PBMC samples were rather increased for the pancreatic cancer patients, compared to the normal persons, with an insignificant difference therebetween. From the data, it was identified that the expression level of IL-29 was significantly increased in pancreatic cancer patients, compared to normal persons, and the expression pattern of IL-29 in pancreatic cancer patients distinctively differed from buffy coat samples to PBMC samples.

[0093] FIG. 2 is a graph showing expression level differences of IL-28 in buffy coat samples obtained from 51 pancreatic cancer patients and 43 normal persons and in PBMC samples obtained from 50 pancreatic cancer patients and 19 normal persons. As can be seen in FIG. 2, the average ΔCt value of IL-28 was significantly reduced in the buffy coat samples from the pancreatic cancer patients, compared to the normal persons. In contrast, the average ΔCt value of IL-28 in the PBMC samples were rather increased for the pancreatic cancer patients, compared to the normal persons, with an insignificant difference therebetween. From the data, it was identified that the expression level of IL-28 was significantly increased in pancreatic cancer patients, compared to normal persons, and the expression pattern of IL-28 in pancreatic cancer patients distinctively differed from buffy coat samples to PBMC samples.

[0094] Taken together, the data indicate that IL-29 and IL-28 are both useful as markers for pancreatic cancer due to their significant difference in expression level between pancreatic cancer patients and normal persons and that when the markers are used, more reliable results could be obtained from a buffy coat sample than a PBMC sample. In addition, a greater difference between pancreatic cancer patients and normal persons was observed for IL-28 than IL-29.

Example 4: Comparison Between Buffy Coat and PBMC from Same Person

[0095] From the same persons (three pancreatic cancer patients (P.C)), buffy coat samples and PBMC samples were prepared referring to Examples 1.1 and 1.2, respectively. The prepared buffy coat and PBMC samples were measured for mRNA expression levels (ΔCt) of IL-29 and IL-28 in the same manner as in Example 2.

[0096] Average mRNA expression levels of IL-29 and IL-28 are depicted in FIG. 4 (No. of samples: three buffy coat samples; three PBMC samples). As shown in FIG. 4, the mRNA expression levels (ΔCt) of IL-29 and IL-28 were significantly higher in the buffy coat samples, compared to the PBMC samples,

[0097] Comparison of mRNA expression levels in buffy coat samples and PBMC samples from same persons were made between pancreatic cancer patients and normal persons and the results are depicted in FIGS. 5a, 5b, 6a, 6b, 7a, and 7b (all expressed as average values).

[0098] FIG. 5a shows IL-28 expression levels in buffy coat and PBMC samples from pancreatic cancer patients in comparison with that in the PBMC samples from normal persons; and FIG. 5b shows IL-28 expression levels in buffy coat and PBMC samples from pancreatic cancer patients in comparison with that in the buffy coat samples from normal persons (no. of samples: 43 normal person buffy coat samples; 3 pancreatic cancer buffy coat samples; 19 normal person PBMC samples; and 3 pancreatic cancer PBMC samples).

[0099] FIG. 6a shows IL-29 expression levels in buffy coat and PBMC samples from pancreatic cancer patients in comparison with that in the PBMC samples from normal persons; and FIG. 6b shows IL-29 expression levels in buffy coat and PBMC samples from pancreatic cancer patients in comparison with that in the buffy coat samples from normal persons (no. of samples: 44 normal person buffy coat samples; 3 pancreatic cancer buffy coat samples; 19 normal person PBMC samples; and 3 pancreatic cancer PBMC samples).

[0100] FIG. 7a shows IL-28 expression levels in buffy coat and PBMC samples from pancreatic cancer patients in comparison with that in the samples from normal persons (average value of integrated IL-28 expression levels in both PBMC and buffy coat samples); and FIG. 7b shows IL-29 expression levels in buffy coat and PBMC samples from pancreatic cancer patients in comparison with that in the samples from normal persons (average value of integrated IL-29 expression levels in both PBMC and buffy coat samples) (no. of samples: 43 and 44 normal person buffy coat samples (for IL-28 and IL-29, respectively); 3 pancreatic cancer buffy coat samples; 19 normal person PBMC samples; and 3 pancreatic cancer PBMC samples).

[0101] As shown in FIGS. 5a, 5b, 6a, 6b, 7a, and 7b, no significant differences of IL-28 and IL-29 expression levels in PBMC samples were not observed between normal persons and pancreatic cancer patients. In contrast, a remarkable difference of IL-28 and/or IL-29 expression level in the buffy coat samples was detected between normal persons and pancreatic cancer patients. These results imply that that pancreatic cancer patients can be diagnosed more accurately and efficiently by measuring the levels of IL-28 and/or IL-29 in buffy coat samples.