JP5407042B2 - Use of seselnin-1, method for predicting prognosis of synovial sarcoma, and reagent kit for testing - Google Patents

Description

  The present invention relates to the use of seselnin-1, a prognostic method for synovial sarcoma using seselnin-1, and a test reagent kit for predicting the prognosis of a patient suffering from synovial sarcoma.

  Synovial sarcoma is a malignant soft tissue tumor that frequently occurs near the limb joints. Histologically, it is a biphasic type consisting of epithelial cells and spindle cells and a monophasic type consisting of spindle cells. are categorized.

  Synovial sarcoma is generally highly malignant and is the most frequently occurring soft tissue tumor after malignant fibrous histiocytoma, liposarcoma, and rhabdomyosarcoma. There are reports that the 5-year survival rate is 30-50%, the 10-year survival rate is 15-30%, and there are cases of local recurrence and metastasis that occur after treatment, and long-term follow-up is required. Tumor.

  Synovial sarcoma is slightly more common among men, although there is no significant gender difference in the incidence between men and women. As the age of occurrence, it occurs frequently in the 10s and 40s, and is particularly likely to occur in young people in their 10s and 20s who are relatively young. As for the occurrence site, the thigh and knee joints are mainly found in the lower limbs and often occur in contact with the joint capsule, bursa, tendon sheath, tendon, etc., but the neck, trunk, etc. not related to these tissues May also occur.

  Synovial sarcoma is painful in about half of patients and the others are asymptomatic, but the tumor grows slowly. Tumors vary from relatively soft to hard.

  Treatment is extensive resection (surgery to remove the tumor by wrapping it in normal tissue as widely as possible), but amputation is indicated for cases that occur near the large joint. Moreover, since it often occurs around nerves, blood vessels and tendons of important muscles, these often have to be removed together, and surgery for reconstructing the function is required at the same time.

  In addition to the possibility of distant metastasis, in addition to surgical excision of the tumor, adjuvant chemotherapy is usually used for treatment, and three- or four-drug combination therapy is generally attempted. .

  There are two types of synovial sarcoma: a poor prognosis group that proliferates malignantly and a good prognosis group that regresses spontaneously, although the mechanism is unknown. Since synovial sarcoma often has a slow local recurrence and metastasis, it is common to require a long-term follow-up of at least 10 years after surgery, but more accurately if the prognosis can be accurately predicted Can follow up treatment policy.

  In addition, if the prognosis can be accurately predicted, unnecessary radiotherapy and chemotherapy can be avoided. Synovial sarcoma cells, in particular, have low sensitivity to anticancer drugs, and have not yet been shown to be effective even with the above-mentioned chemotherapy combination therapy, while chemotherapy shows a response at a certain rate. It has also been reported to cause serious side effects. Therefore, if a case with good prognosis or poor prognosis can be identified in advance, the benefit of avoiding unnecessary chemotherapy is great, and contributes to the patient's quality of life (QOL).

  In synovial sarcoma, chromosomal translocation t (X; 18) (p11.2; q11.2) has been reported to occur frequently. Isolation of genomic DNA in the vicinity of the breakpoint fused the SYT gene with t (X; 18) located on chromosome 18q11.2 and the SSX gene located on chromosome Xp11.2, thereby expressing the chimeric protein SYT-SSX It has become clear to do. Since SYT-SSX expression has been observed in almost all synovial sarcoma cases, this is thought to play a central role in tumor development. Patent Document 1 describes that there is a certain degree of relationship between SYT-SSX1 or SYT-SSX2 fusion and tumor morphology and 5-year survival rate.

  Non-Patent Document 1 also analyzed SYT-SSX fusion transcripts of 45 synovial sarcomas (33 monophasic and 12 biphasic) by reverse transcription polymerase chain reaction, and related clinical and pathological data. As a result of comparison, SYT-SSX1 and SYT-SSX2 fusion transcripts were detected in 29 (64%) and 16 (36%) tumors, respectively, and analyzed by Kaplan-Meier method of 39 patients with local tumors. So, 15 patients with SYT-SSX2 may show significantly better metastasis-free survival than 24 patients with SYT-SSX1 (P = 0.03 by multivariate analysis; relative risk of 3.0). Have been described.

  In Non-Patent Document 2, when SYT-SSX1 / SSX2 fusion gene was searched for 33 frozen material samples, SYT-SSX1 was significantly worse prognostic factor (5-year metastasis-free survival than SYT-SSX2). Rate: SYT-SSX1 42% VS SYT-SSX2 89%).

Japanese Patent Laid-Open No. 2003-252802

The New England Journal of Medicine 1998; 338: 153-60, Kawai Akira Synovial sarcoma. A Scandinavian Sarcoma Group project, Acta Orthop Scand Suppl / 291, 1-28 / 2000, Skytting B

  The above-mentioned translocation gene SYT-SSX is known to be specifically expressed in 90% or more of synovial sarcomas, but may not be expressed.

  An object of the present invention is to provide a novel and accurate method for predicting the prognosis of synovial sarcoma, and to provide use of seselnin-1 that can easily and accurately predict the prognosis of synovial sarcoma.

  The use of seselnin-1 according to the first aspect of the present invention is a use as a biomarker for predicting the prognosis of synovial sarcoma.

  The prognosis is, for example, after treatment including at least one of surgery, chemotherapy, radiation therapy, hyperthermia, and immunotherapy.

  The method for predicting the prognosis of synovial sarcoma according to the second aspect of the present invention measures the expression level of seselnin-1 and, when the expression level of seselnin-1 is increased, the prognosis for synovial sarcoma is good. It is characterized by prediction.

  The expression level of selcernin-1 is preferably measured by an immunological method using a selcernin-1-specific antibody.

  The immunological method is, for example, any method selected from Western blotting, immunoprecipitation, dot blotting, slot blotting, and ELISA.

  The prognosis is, for example, after treatment including at least one of surgery, chemotherapy, radiation therapy, hyperthermia, and immunotherapy.

  A test reagent kit according to a third aspect of the present invention is a kit for predicting the prognosis of a patient suffering from synovial sarcoma, wherein the expression level of sesernin-1 in a sample obtained from the patient is detected or detected. It includes means for quantifying.

  According to the present invention, the prognosis of synovial sarcoma can be predicted easily and accurately. Therefore, it is possible to shorten the follow-up treatment period by accurately establishing a post-operative treatment policy, and to enable precise and optimized individualized medicine. In addition, unnecessary radiotherapy and chemotherapy can be avoided, and further, it is useful for development of a novel treatment method for synovial sarcoma.

The process of 2D-DIGE method is shown. Fig. 3 shows detection of protein spots in an electrophoresis experiment. The reproducibility of the electrophoresis separation experiment is shown. The correspondence between protein spots and protein names is shown. The result of principal component analysis is shown. Shows the log rank test for survival. A log rank test for the metastasis-free rate is shown.

  In the present embodiment, the expression level of the patient's selernin-1 (secernin-1) is measured. There are 1 to 3 types of selcernin. In the present invention, attention is paid to the expression level of selcernin-1.

  And when the expression level of seselnin-1 is rising, it is estimated that the prognosis with respect to synovial sarcoma is favorable.

  The measurement of the expression level of selcernin-1 is not particularly limited, and it may be simply detecting the presence or absence of selcernin-1, and the expression level of selcernin-1 is determined relative or absolute. It may be a thing. The expression level of seselnin-1 is preferably measured by an immunological technique. For example, a combination of separation by electrophoresis and detection or quantification by fluorescence, enzyme, radioisotope, etc. (Western blotting, fluorescence 2D electrophoresis method), immunostaining method (fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, including radioisotope labeled antibody method), enzyme immunoassay adsorption method (ELISA), dot blotting method, etc. Can be performed. In the present embodiment, a living tissue is collected from a patient, a protein contained in the living tissue is subjected to electrophoresis, and the presence and amount of seselnin-1 is measured with a fluorescent dye.

  The treatment that can predict the prognosis is not particularly limited, and is, for example, surgery, chemotherapy, radiation therapy, hyperthermia, or immunotherapy.

  The surgical procedure for synovial sarcoma is usually extensive resection. This is because in synovial sarcoma, tumor cells are soaked around a tumor that can be visually confirmed, and it is necessary to excise a large amount of tissue including normal surrounding tissue. In addition, because extensive resection has been performed, if there is a large amount of skin, chemotherapy and radiation therapy cannot be performed. Therefore, it is necessary to perform flap surgery to bring skin with blood vessels from other parts of the body. .

  In general, three-drug combination therapy or four-drug combination therapy has been tried as chemotherapy for synovial sarcoma. A typical example of triple combination therapy is, for example, MAID therapy (Mesna: side effect reducing agent, Adriacin: adriamycin, Ifomide: ifosfamide, Dacarbazine: dacarbazine). A representative example of the four-drug combination therapy is, for example, CyVADIC therapy (Cyclophosphamide: cyclophosphamide, Vincristine: vincristine, Adriacin: adriamycin, dacarbazine: dacarbazine).

  As with general radiotherapy, synovial sarcoma radiotherapy differs in biological response (tolerated dose) even with the same irradiation dose depending on the size of the irradiation volume. Whether it is used together or before or after surgery is controlled by the judgment of the treating physician. The dose required for tumor control depends on the sensitivity of the tumor, and the dose is, for example, 60-70 Gy. As the dose, divided irradiation in which a small dose is irradiated once a day and 4 to 5 times a week is often performed. In the case of divided irradiation, the amount of one line is, for example, 1.8 to 2 Gy.

  Hyperthermia is a treatment aimed at raising the internal temperature of a tumor by applying heat and killing cancer. The applied temperature is, for example, about 43 ° C. Compared with normal cells in which blood vessels dilate and increase blood flow when heat is applied, tumor cells do not dilate the blood vessels, so heat accumulates. Cheap.

  Immunotherapy is a treatment method that improves the action of the body's natural defense mechanisms against diseases, and is usually treated by activating the local or systemic immune system. Specifically, immunotherapy using lymphocytes having an antitumor effect, cancer vaccine therapy, cancer vaccine tailor-made treatment, DNA vaccine therapy, RNA vaccine therapy, and the like.

  In the present invention, a favorable prognosis means that there is a statistically significant difference in metastasis-free rate or survival rate in a period of 5 years or longer.

  The kit according to the present embodiment is a test reagent kit for predicting the prognosis of a patient suffering from synovial sarcoma, and includes means for detecting or quantifying the expression level of seselnin-1 in a sample obtained from the patient. When the test is performed by an immunological technique, at least anti-sesernin-1 antibody is included in the test reagent. The anti-sesernin-1 antibody is not particularly limited as long as it can detect the expression of seselnin-1, and for example, monoclonal and polyclonal antibodies, labeled antibodies, chimeric antibodies, humanized antibodies, and binding activity fragments thereof. Is mentioned. Moreover, the test reagent kit may contain a label used for detection in addition to the antibody. The kit can contain reagents such as a buffer, a chromogenic substrate, a secondary antibody, and a blocking agent.

  In synovial sarcoma, a biopsy is usually performed with a primary tissue for the purpose of histopathological diagnosis. If a biological tissue is collected at that time, a new burden is not imposed on the patient.

  Moreover, according to the invention which concerns on this embodiment, since the expression level of seselnin-1 can be measured, for example, if there is only a Western blot apparatus, the prognosis of synovial sarcoma can be predicted by a simple technique. For example, if an antibody specifically expressed in seselnin-1 is used, the expression level of sesernin-1 can be measured with sensitivity by methods other than Western blotting, such as immunostaining.

Example 1
<Patient and clinical information>
Thirteen frozen tumor tissues obtained from patients with synovial sarcoma were examined. Frozen tumor tissue was obtained in 13 synovial sarcoma cases and used in proteomic analysis. Diagnosis and classification was based on the WHO classification system for soft tissue tumors. SYT-SSX1 and SYT-SSX2 expression were also examined. The results are shown in Table 1 below.

  As shown in Table 1, synovial sarcoma samples were divided into two groups. That is, five samples collected from patients who died of poor prognosis of synovial sarcoma (P-SS) (sample numbers are 1,2,3,4,5) and good prognosis of synovial sarcoma ( G-SS) was classified into 8 samples (sample numbers were 6, 7, 8, 9, 10, 11, 12, 13) that had no metastasis within 5 years after surgery. DOD (dead of disease) means disease death, AWD (alive with disease) means survival in disease state, NED (no evidence disease) means survival without disease confirmation, and CDF (continuous disease free) means disease-free survival. .

<Profiling protein expression>
The frozen sample was cooled with liquid nitrogen and then pulverized into a powder by a cryopress (registered trademark, Microtech Nichion, Chiba, Japan). Frozen samples were prepared with urea lysis buffer (6M urea, 2M thiourea, 3% CHAPS, 1% Triton X-100). After centrifugation at 15,000 rpm for 30 minutes, the supernatant was removed and used as a source of cellular proteins.

  Thereafter, electrophoresis separation was performed by 2D-DIGE (2 Dimensional Fluorescence Difference Gel Electrophoresis). The 2D-DIGE method is a method in which samples to be compared before electrophoresis are labeled with different fluorescent dyes, and the labeled samples are mixed and then subjected to electrophoresis. FIG. 1 is a process diagram showing processes of the 2D-DIGE method. The control sample (internal control sample) was prepared by mixing a part of all biopsy specimens, and 5 μg was used. The control sample was labeled with Cy3 (registered trademark, CyDye DIGE Fluor saturation dye, GE Healthcare Biosciences, Uppsala, Sweden) as shown in FIG. Each biopsy specimen was used in an amount of 5 μg and labeled with Cy5 (registered trademark, CyDye DIGE Fluor saturation dye, GE Healthcare Biosciences, Uppsala, Sweden).

  Primary separation was performed on IPG buffer and DryStrip gel (24 cm length, pI range between 4 and 7, GE Healthcare Biosciences). Secondary separation was performed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE: EttanDalt II GE Healthcare Biosciences) using a large gel (38 cm length, Bio-craft, Itabashi, Tokyo, Japan). It was. The gel was scanned by laser irradiation (Tyhoon Trio®, GE Healthcare Biosciences). In all spots, Cy5 intensity was initialized by Cy3 intensity, and differences between gels were corrected by DeCyder image software (GE Healthcare Biosciences). The results are shown in FIG.

  FIG. 3 shows the reproducibility of the electrophoresis separation experiment. As shown in FIG. 3, it was demonstrated that reproducibility exists by comparing three independent electrophoretic separation experiments (exp1, exp2, exp3) for the same sample. According to the scatter plot analysis, the standard intensity of 96% or more of the spots was within the range of 2 fold difference.

<Data analysis>
The intensity of the protein spot was statistically significantly different between the two groups (Wilcoxon test P value <0.01). Hierarchical summarization, principal component analysis, correlation matrix surveys, and spot ranking were performed using expression software (Genedata, Bersel, Switzerland).

<Protein identification by mass spectrometry>
1663 protein spots appearing in at least 75% of the control sample image labeled with Cy3 were selected. Proteins corresponding to protein spots were identified by mass spectrometry. Proteins labeled with Cy5 separated by 2D-PAGE were recovered in gel plugs and assembled with modified trypsin (Promega, Madison, Wis.). Mass spectrometry used a Finnigan® LTQ® linear ion trap mass spectrometer (Thermo Electron, San Jose, Calif.) With a nanoelectrospray ion source (AMR, Tokyo). Mascot software (version 2.1, Matrix Science, London, UK) was used to perform mass surveys of peptide ion peaks against the SWISS-PROT database (Homo sapiens, 12867 sequence in Sprot 47.8 fasta file). A mascot score of 35 or higher was considered indicative of positive protein identification. The results are shown in FIG. Twenty protein spots had significant differences between the two groups (P <0.01).

<Protein classification>
Moreover, the functional classification of the identified protein was based on the classification of gene ontology as shown in Table 2 below.

  As a result of protein identification using a mass spectrometer, as shown in Table 2 and FIG. 4, three protein spots (sesernin-1 spot) derived from a protein called seselnin-1 are included in 20 spots. It was. The intensity of 12 protein spots decreased with poor prognosis (P-SS), and the intensity of 8 protein spots increased with poor prognosis (P-SS).

  When the expression of cesernin-1 was examined on 13 frozen tumor tissues obtained from patients with synovial sarcoma, the concentration of cesernin-1 spot was low expressed in the 5 cases with the above-mentioned poor prognosis, It was highly expressed in 8 cases with good prognosis.

  Thirteen synovial sarcoma samples were accurately classified into good prognosis (G-SS) or poor prognosis (P-SS) groups by principal component analysis. Principal component analysis is a method of constructing new variables expressed by linear combinations from data statistics expressed in multivariate and summarizing them into uncorrelated principal components. As shown in FIG. 5, the profile of the 13 protein spots was similar between samples of the same group, but different between the two groups. From the above, it was shown that seselnin-1 can be used as a biomarker for predicting the prognosis of synovial sarcoma.

(Example 2)
For the purpose of further verifying the prognostic ability of seselnin-1, a specific antibody against seselnin-1 was newly prepared, and immunostaining was performed on 45 cases.

<Production of antibodies>
A peptide corresponding to peptide length 354-370 of full-length seselnin-1 was synthesized and rabbits were immunized. The antiserum was purified by affinity column chromatography using the antigenic peptide. The specificity of seselnin-1 was confirmed by Western blot.

  45 paraffin specimen tissues were examined from 45 patients prior to treatment. The clinical information of patients used in immunohistochemistry is summarized in Table 3 and Table 4 below.

<Immunohistochemistry for seselnin-1>
Seselnin-1 expression was examined immunohistochemically using paraffin specimen tissue. 4 μm thick tissue sections were autoclaved in 10 mM citrate buffer (pH 6.0) at 121 ° C. for 30 minutes and cultured with antibodies against seselnin-1 (1: 200 dilution). Immunostaining was performed by labeling with biotin-free horseradish peroxidase from the DACO Envision System (Envision Plus label: DAKO, CA). Two certified pathologists (NT and DK) determined positive staining by clinical data (age, gender, anatomical results). Cases of 20% or more of the tumor cells positive for the anti-sesernin-1 antibody were considered to be seselnin-1 positive, and cases of 20% or less of the sesernin-1 positive were considered to be sesernin-1 negative.

<Molecular analysis>
Of the 45 cases, 31 cases where RNA samples were available were investigated for the fusion gene of SYT-SSX1 and SYT-SSX2. Reverse transcription polymerase chain reaction (RT-PCR) using primers SYT 5′CAA CAG CAA GAT GCA TAC CA3 ′, SSX1 5′GGT GCA GTT GTT TCC CAT CG3 ′, and SSX2 5′GGC ACA GCT CTT TCC CAT CA3 ′ All 31 cases were analyzed.

  As shown in the above Table 3 and Table 4, out of 45 cases, seselnin-1 positive cases are 27 cases (sample numbers are 3, 4, 9, 10, 11, 17, 20, 21, 22, 24, 25, 27,29,30,32,33,34,35,36,38,39,40,41,42,43,44,45), 18 cases of selcernin-1 negative cases (sample numbers are 1,2, 5,6,7,8,12,13,14,15,16,18,19,23,26,28,31,37). In these cases, the correlation between metastasis after the start of treatment and the expression of seselnin-1 was examined. Clinical information on metastasis after the start of treatment was obtained in all 45 cases. Among 45 patients, 15 patients continue to survive without disease (continuous disease free (CDF)), 5 patients survive without evidence of disease (NED), 3 Of the patients were alive with disease (AWD), and 22 patients were dead of disease (DOD).

As shown in Table 5 below, metastasis was 12 out of 27 cases (44.4%) in the seselnin-1 positive cases and 15 out of 18 cases (83.3%) in the negative cases. An extremely high rate of metastases was observed in patients with selcernin-1-negative tumors compared to those with selcernin-1-positive tumors (15/18 vs 12/27 cases, χ ² : P = 0.0140).

Moreover, as shown in Table 6 below, deaths were 9 cases (33.3%) in 27 cases in the seselnin-1 positive cases, and 13 cases (72.2%) in 18 cases in the negative cases. By immunohistochemistry, 5 of the 23 surviving patients were selcernin-1 negative and 18 of the 23 surviving patients were sesernin-1 positive (χ ² : P = 0.0170).

  Next, the relationship between the expression of seselnin-1 and the metastasis-free rate and survival rate for 5 years after the start of treatment was examined.

<Statistical analysis>
From the first treatment of the initial tumor to the death due to the presence of a tumor specific cause or metastasis, the duration of survival without tumor specific and metastasis was calculated. Survival rate for each death was calculated by Kaplan-Meier method. Univariate analysis using the log rank test identified potential prognostic factors. Independent prognostic factors were assessed using the Cox proportional hazards model and the stepwise method. Covariance analysis was chosen in the model only if it contributed significantly to model matching based on chi-square test values. A P difference <0.05 was considered important. Statistical analysis was performed using the SPSS statistical package (SPSS, Chicago, Illinois).

  FIG. 6 shows the result of the log rank test for the survival rate. As shown in Table 7 and FIG. 6 below, the 5-year survival rate of patients positive for selcernin-1 immunoreactivity is 77.6% and 5% of patients negative for selsernin-1 immunoreactivity. The annual survival rate was 21.8% (P = 0.0015).

  FIG. 7 shows the result of the log rank test for the non-transfer rate. As shown in Table 7 and FIG. 7 below, the metastasis-free rate was 62.8% in the positive case of seselnin-1 expression and 16.7% in the negative case. A very high proportion of patients with seselnin-1 positive tumors compared to those with seselnin-1 negative tumors were observed to survive 5 years without metastasis (62.8% vs 16.7%; P = 0.0012).

  From the above, the presence / absence of selsernin-1 expression has a statistically significant correlation with the prognosis of synovial sarcoma, and it is demonstrated that selcernin-1 is useful as a prognostic biomarker in the diagnosis of synovial sarcoma. It was. In clinical practice, by examining the expression of seselnin-1 at the time of diagnosis of synovial sarcoma, it is possible to accurately predict the prognosis of patients with synovial sarcoma, and to select an appropriate treatment method such as the presence or absence of adjuvant chemotherapy, It enables precise and optimized personalized medicine. Since soft tissue sarcomas including synovial sarcomas are always biopsied from the primary tissue for the purpose of histopathological diagnosis, the biopsy material used in this examination can be collected without placing a new burden on the patient. Immunostaining is a routine test. Therefore, the prognosis prediction of synovial sarcoma according to the present invention can be easily put into practical use as a clinical test, and many synovial sarcoma patients can greatly benefit from the present invention.

  Sequence number 1-3: Primer

Claims (7)

Use of seserin-1 as a biomarker to predict the prognosis of synovial sarcoma. The use of seselnin-1 according to claim 1, wherein the prognosis is after treatment including at least one of surgery, chemotherapy, radiation therapy, hyperthermia, and immunotherapy. Prognosis prediction of synovial sarcoma characterized by measuring the expression level of sesernin-1 (secernin-1) and predicting that the prognosis for synovial sarcoma is good when the expression level of sesernin-1 is increased Method. The method for predicting prognosis of synovial sarcoma according to claim 3, wherein the expression level of selcernin-1 is measured by an immunological method using a selcernin-1-specific antibody. The immunological method is Western blotting, fluorescence two-dimensional electrophoresis, fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, radioisotope labeled antibody method, enzyme immunoassay adsorption method (ELISA), or dot The method for predicting prognosis of synovial sarcoma according to claim 4, wherein the method is any method selected from blotting methods. 6. The slip according to claim 3, wherein the prognosis is after treatment including at least one of surgery, chemotherapy, radiation therapy, hyperthermia, and immunotherapy. Prognosis prediction method of membranous sarcoma. A test reagent kit for predicting the prognosis of a patient suffering from synovial sarcoma, comprising a means for detecting or quantifying the expression level of secernin-1 in a sample obtained from the patient.

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