WO2015108195A1 - Method for evaluating malignancy of malignant tumor in non-human animal - Google Patents
Method for evaluating malignancy of malignant tumor in non-human animal Download PDFInfo
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- WO2015108195A1 WO2015108195A1 PCT/JP2015/051290 JP2015051290W WO2015108195A1 WO 2015108195 A1 WO2015108195 A1 WO 2015108195A1 JP 2015051290 W JP2015051290 W JP 2015051290W WO 2015108195 A1 WO2015108195 A1 WO 2015108195A1
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
Definitions
- the present invention relates to a method for evaluating the malignancy of a malignant tumor of a non-human animal. More specifically, the method for evaluating the malignancy of a malignant tumor of a non-human animal of the invention includes a method for predicting the prognosis of the tumor.
- Mast cell tumors are tumors that are common in dogs and cats and often occur in the skin. In dogs, they are the most common skin tumor and the second most common in cats. .
- Canine cutaneous mastocytoma is one of the common skin tumors, accounting for 7-21% of all skin tumors, and its biological behavior is very diverse. While there are benign cases that can be completely cured by surgical resection, there are various types of cases such as those that invade regional lymph nodes, those that recur after surgical resection, and those that cause fatal metastases Are known. Therefore, an index of malignancy of canine cutaneous mastocytoma or an index for predicting prognosis has been required.
- Patnaik's classification method proposed by Patnaik et al. Has been used. Patnaik's classification method is based on the extent of lesion, cell density, cell morphology, fission index, stromal response, etc., well differentiated (grade I), moderately differentiated (grade II), poorly differentiated (grade) Classify as III).
- the mitotic index is one of the most important indices (Non-patent Document 3).
- the present inventors examined a method for more accurately evaluating the malignancy of a non-human animal malignant tumor and predicting the prognosis. That is, the present invention relates to a method for evaluating the malignancy of a malignant tumor of a non-human animal and predicting a prognosis.
- CD34 is a transmembrane protein and is usually expressed in skeletal muscle satellite cells, gastrointestinal chahar-mediated cells, vascular endothelium, brain neuronal cell bodies, and dermal dendritic cells. CD34 is also expressed in hematopoietic stem cells, and CD34 positive cells are known as mast cell progenitors (Kawarai et al., J Vet Med Sci.
- CD34 may have different expression depending on the degree of differentiation of tumor cells of cutaneous mastocytoma.
- bone marrow-derived cultured mast cells obtained from CD34-deficient mice have also been reported to aggregate with each other compared to mast cells obtained from wild-type mice (Drew E, et al. , Immunity. 2005 Jan; 22 (1): 43-57.). Therefore, as a result of examining the possibility as a new prognosis or metastasis factor, the inventors have found that the level of immunostaining with an anti-CD34 antibody in a sample derived from canine cutaneous mastocytoma is not less than +2 (described later). It has been found for the first time that the prognosis is poor when the proportion of cells is approximately 20% or more.
- the present invention includes the following (1) to (11).
- a method for evaluating the malignancy of a tumor in a non-human animal wherein CD34 expressed in cells in a sample derived from a subject animal containing tumor cells is detected, and the CD34 against the cells present in the sample is detected. The ratio of the cell which expresses is calculated, The method of evaluating the malignancy of this tumor based on the ratio.
- a diagnostic kit for evaluating the malignancy of a tumor of a non-human animal comprising an anti-CD34 antibody.
- the non-human animal is a dog or a cat, and the tumor is cutaneous mastocytoma.
- the malignancy evaluation method and prognosis prediction method of the present invention it is possible to accurately diagnose the malignancy and prognosis of tumor cells of non-human animals, particularly mastocytoma.
- the malignancy evaluation method and prognosis prediction method of the present invention can provide a clue for determining the resection range of a tumor.
- the present invention Until now, unless surgery was performed, malignancy or prognosis could not be determined, and it was necessary to consider the indication of chemotherapy in all cases. According to the present invention, the possibility of metastasis can be predicted without performing image diagnosis, and chemotherapy can be started in accordance with the results of the present invention. That is, it is possible to perform appropriate chemotherapy for necessary cases. In this respect, the present invention also exerts an effect that it is not necessary to perform unnecessary chemotherapy for a negative case.
- FIG. 1 is an ROC curve for canine mastocytoma.
- FIG. 2 is a diagram showing the relationship between Patnaik grade and CD34 staining according to the present invention.
- FIG. 3 shows the results of immunostaining of cells derived from canine mastocytoma with anti-CD34 antibody. Refer to the text of the specification for staining 1+, 2+ and staining 3+.
- FIG. 4 shows the log rank test results regarding the presence or absence of metastasis of mastocytoma in dogs and the number of days of survival.
- FIG. 5 shows the log rank test results regarding the staining ability of CD34 antibody-derived cells of canine mastocytoma-derived cells and the survival days.
- the present invention is a method for evaluating the malignancy of a tumor cell, predicting the prognosis, or the presence of metastasis using the expression level of CD34 expressed in the tumor cell as an index. That is, one of the embodiments of the present invention is a method for evaluating the malignancy of a tumor in a non-human animal or predicting prognosis, and is expressed in cells in a sample derived from a target animal including tumor cells. When the expression of CD34 is detected and the number of cells expressing CD34 is a certain percentage or more of the total number of cells present in the sample, it is judged that the tumor has a high malignancy or a poor prognosis Is the method.
- “high malignancy” means that the prognosis thereafter is poor, and the malignancy obtained using the tumor malignancy evaluation method of the present invention is determined by surgical operation of the tumor. This is effective in determining the range of resection, and the choice of treatment methods such as the necessity of pre- or post-operative adjuvant therapy or medical therapy (such as chemotherapy using chemotherapeutic agents or molecular targeted drugs). Accordingly, the use of the present invention to determine the therapeutic range and method of treatment of the tumor naturally falls within the scope of the present invention within the scope of the present invention.
- “prognosis” has the same meaning as used in the medical field and is not particularly limited.
- a view regarding an expected medical condition (health condition), a future of illness / wound It is a state.
- the prognostic evaluation or prognosis includes, for example, grade diagnosis of cancer, malignancy diagnosis, prediction of the presence or absence of metastasis in the future, presence or absence of deterioration of symptoms after surgery Predictions can be mentioned.
- the poor prognosis refers to, for example, a case where there is a possibility that the survival rate is shortened, the risk of recurrence is increased, and / or the tumor has metastasized to another site.
- the tumor cells in the present invention are cells that originate from cells expressing CD34, such as cells derived from hair follicle stem cells, mast cells, vascular endothelial cells, fibroblasts, and Langerhans cells. Particularly preferred are mastocytoma cells.
- the non-human animal that is the subject of the prognostic evaluation method of the present invention is not particularly limited, and examples thereof include dogs, cats, ferrets, and the like.
- CD34 is a 110 kDa single-chain transmembrane phosphorylated glycoprotein, but when described herein as “CD34”, it refers to a protein.
- Nucleic acid Information such as the amino acid sequence and nucleic acid sequence of CD34 has already been disclosed in public databases, and can be easily obtained by those skilled in the art.
- the canine CD34 amino acid sequence and nucleic acid sequence are SEQ ID NOs: 1 and 2, respectively
- the feline CD34 amino acid sequence and nucleic acid sequence are SEQ ID NOs: 3 and 4, respectively.
- a preferred embodiment of the present invention includes the step of detecting CD34.
- means for collecting a sample of tumor cells from the animal to be diagnosed may be any method easily selected by those skilled in the art, for example, a needle biopsy for obtaining a cell sample using a puncture needle, or a surgical incision. It can be carried out by a method such as an incision biopsy to obtain an affected tissue piece.
- the expression state of CD34 in a diagnostic sample containing tumor cells can be carried out by a method that can be easily selected by those skilled in the art.
- a tissue specimen or cytological specimen can be prepared and examined.
- the tissue specimen or cell specimen may be produced using any known method.
- the collected tissue or the like can be fixed with formalin or the like, and after paraffin embedding treatment, a section can be prepared and immunohistochemical staining can be performed to examine the expression level of CD34.
- it can also implement by Liquid Based Cytology (LBC: Liquefaction cytology) which is one method of cytodiagnosis.
- LBC Liquid Based Cytology
- an antibody against CD34 can be used to detect CD34 expressed in collected tissues or cells by an immunohistochemical technique.
- anti-CD34 antibody any one of an antibody prepared by a person who carries out the present invention or a commercially available antibody (for example, SANTARUCRUZ BIOTECHNOLOGY, # sc-7045) can be used. Or a polyclonal antibody may be sufficient.
- the antibody does not have to be a complete antibody, and may be a fragment containing a CDR region or the like, or may be prepared by genetic engineering.
- the antibody fragment may be any fragment that binds to CD34 expressed on cells and can be used for immunohistochemical staining.
- DAB diaminobenzidine
- ACE aminomethylcarbazole
- BCIP / NBT 5-bromo-4-chloro-3-indoxyl Phosphate / nitro blue tetrazolium chloride
- a tissue or cell specimen stained at a low magnification is observed with a microscope, a region having the strongest staining intensity is selected, and then the region is observed under a high-magnification visual field to select 100 cells to be observed.
- the staining intensity of each of the selected 100 cells is classified into the following four (see also FIG. 1). 0: Cells that are not stained or are slightly stained as much as the background. 1+: Cells that are indistinguishable from 0 at a low magnification, but are light and stainable at a high magnification. 2+: Cells whose staining property can be confirmed at a low magnification and completely stained at a high magnification. 3+: Cells that are completely stained at low magnification.
- Cells having a staining ability of 2+ or more with respect to 100 selected cells are determined as “cells expressing CD34”, and the ratio is calculated.
- the ratio is 10% or more, 15% or more, more preferably 20% or more.
- the above four levels of staining may be evaluated by quantifying and digitizing the staining intensity obtained from the image.
- a high-magnification field image (200 to 400 times) of a main tumor cell group stained on a microscope is photographed, and the color stained with CD34 expression positive in the photographed image is displayed as a bioimaging analysis system (Lumina Vision, Mitani Corp.) and divide it into red, green, and blue RGB colors.
- the stained positive area on the photographed image is obtained by subtracting the negative area that is non-specifically stained with the negative antibody, and the ROC curve is obtained by statistical analysis between the positive area and the metastasis and survival results to cut off.
- a value can be set and a cutoff value or higher can be determined as positive.
- CD34 mRNA expressed in tumor cells in a sample may be detected by a so-called hybridization method to monitor the expression status of CD34.
- hybridization methods include in situ hybridization methods (see, for example, Pascucci et al., Vet Dermatol. 2006 Aug; 17 (4): 244-51).
- Labeled probes complementary to CD34 mRNA such as radioactively labeled probes, digoxigenin (DIG) probes, fluorescently labeled (FITC, RITC, etc.) probes, etc. Can be used to detect the amount of CD34 mRNA in a sample section or specimen.
- Evaluation of the amount of mRNA of CD34 in the sample is performed by observing the signal obtained from the labeled probe under a microscope, classifying the observed cells into four stages as described above [0019] based on the signal intensity from the label, The ratio of cells having a signal intensity of 2+ or more can be calculated and used as an indicator of prognosis.
- the amount of CD34 mRNA expressed in a sample is detected by quantitative RT-PCR (real time PCR) as a method for detecting the level of CD34 expressed in tumor cells in the sample. May be.
- Another embodiment of the present invention is a kit for evaluating malignancy of a tumor cell that develops in a non-human animal such as a dog or a cat, for example, a mastocytoma, or a prognosis diagnostic kit.
- the present invention provides a method for evaluating the malignancy of a tumor cell and predicting the prognosis using the expression level of CD34 in the tumor cell as an index. Therefore, the probe or primer used to measure the expression level of anti-CD34 antibody or CD34 mRNA used to measure the expression level of CD34 in tumor cells contained in the sample is the prognosis of non-human animals. This application is disclosed for the first time in the present invention.
- the diagnostic kit for prognosis of tumor cells of the present invention includes a probe for detecting CD34 expressed in cells as an essential component thereof, for example, an anti-CD34 antibody, a probe for detecting CD34 mRNA. It is.
- a fixing agent such as formalin necessary for immunostaining a tissue or cells to be diagnosed, immunohistochemical staining or quantitative RT-PCR is performed.
- it may contain a chromogenic substrate, a buffer and the like.
- Experimental method 1-1 Test animals Visited the department of oncology at Azabu University Hospital between 2007 and 2011. After surgical removal, the tumor was removed and submitted to the pathology laboratory of the hospital to be diagnosed with mastocytoma. Fifteen cases were included. Information about the clinical course, date of collection, age, sex, dog breed, body weight, distribution and number of mass lesions, location of mass, and surgical margin for each case can be obtained from the histopathology request form. It was. Information about the cases is summarized in Table 1.
- the embedded tissue is sliced to 3-5 ⁇ m and then slide glass (MICRO SLIDE GLASS pre-clean water drainage, code S-7224, Matsunami Glass Industry) (MICRO SLIDE GLASS water edge polishing frost, code S-8215, Matsunami Glass Industry) ) To obtain tissue sections, and immunohistochemical staining was performed.
- Hematoxylin and eosin (HE) staining Tissue sections were deparaffinized (xylene I for 7 minutes, xylene II for 7 minutes, 99% alcohol I for 5 minutes, 99% alcohol II for 10 minutes, 80% alcohol for 7 minutes And 70% alcohol for 7 minutes) and passed through distilled water 10 times. Subsequently, the mixture was passed through a hematoxylin solution (Tissue Tech Hematoxylin 3G, code 8656, Sakura Finetech Japan) for 3 minutes, washed with running water for 30 minutes, and distilled water 10 times.
- a hematoxylin solution Tissue Tech Hematoxylin 3G, code 8656, Sakura Finetech Japan
- eosin liquid Tissue Tech eosin, code 8659 Sakura Finetech Japan
- color separation and dehydration treatment 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, each several times, 99% Alcohol I, 99% Alcohol II, 99% Alcohol III, 99% Alcohol IV for 5 minutes each
- thorough treatment xylene I for 7 minutes, xylene II for 7 minutes, xylene III for 10 minutes
- Encapsulant (NEW M • X, code FX00500, Matsunami Glass Industry Co., Ltd. was used.
- Antigen activation treatment was performed using a pressure kettle (Tifar krypsocler, model P4310731, Group Cebu Japan), and the sections were immersed in the antigen activation solution (Dako REALTM Target Retrieval Solution, code S2031, Dako) and pressurized for 5 minutes. . After the pressure treatment, the mixture was cooled at room temperature for 20 minutes, and then washed with PBS.
- the primary antibody is an anti-human CD34 goat polyclonal antibody that is known to cross-react with dogs immunohistochemically (sc-7045, SANTA CRUZ; Jennings et al., Vet Pathol., 49: 532-537, 2012) Was used. After flowing blocking serum, the primary antibody was added dropwise and reacted at a concentration of 1: 100 overnight at 4 ° C. (about 12 hours).
- goat IgG PURIFIED GOAT IgG, code PCP001, AbD SEROTEC
- KITPBS KITPBS was washed to remove excess water.
- a histofine biotin-labeled anti-goat IgG antibody code 416022, Nichirei
- the secondary antibody was reacted at room temperature for 10 minutes.
- histofine peroxidase-labeled streptavidin code 426062, Nichirei
- PBS was washed to remove excess water.
- a simple stain DAB solution (code 415172, Nichirei) was added dropwise, and color development was performed for 7 minutes under a microscope. After stopping color development with distilled water, counterstaining was performed with Mayer's hematoxylin (code 30002, Muto Chemical) for 30 seconds, washed with running water for 30 minutes, and passed through distilled water 10 times. Then, dehydration treatment (70% alcohol, 80% alcohol, 90% alcohol, 99% alcohol I, 99% alcohol II, 99% alcohol III each for 7 minutes) and clearing treatment (xylene I, xylene II, xylene III each 7 minutes) and sealed with M ⁇ X.
- dehydration treatment (70% alcohol, 80% alcohol, 90% alcohol, 99% alcohol I, 99% alcohol II, 99% alcohol III each for 7 minutes
- clearing treatment xylene I, xylene II, xylene III each 7 minutes
- Histopathological evaluation (1) HE staining Grade sections of each case diagnosed as cutaneous mastocytoma were classified according to Patnaik's report (Patnaik et al., Vet Pathol., 21: 469-474, 1984). It was. Observations include tumor invasion site, cell density, morphology of cells and nuclei, cell borders, presence or absence of cytoplasmic granules, fission image in high magnification field, presence or absence of eosinophil infiltration, degree of sweat gland, lymphatic vessel dilation Interstitial reactions including presence and extent, presence or absence of collagen fiber degeneration, necrosis, edema and bleeding were performed.
- the epidermis When evaluating the site of tumor invasion, the epidermis is a layer composed of stratified squamous epithelial cells with a keratinized layer, and the dermis is a dense fiber in the epidermis where hair follicles, sebaceous glands, sweat glands, lymphatic vessels, etc. are observed Sexual connective tissue and subcutaneous tissue were fibrillar connective tissue including adipose tissue. Furthermore, the dermis was divided into three layers, the layer from the epidermis to the region where the appendages were present was the shallow layer, the region where the appendages were present was the middle layer, and the layers below the middle layer were the deep layers. For the subcutaneous tissue, the upper layer close to the dermis was the shallow layer, and the lower layer close to the muscle layer was the deep layer.
- 1+ Indistinguishable from 0 at low magnification, but light and highly stainable at high magnification.
- the prognosis of grade 3 mastocytoma is poor, but when the resection margin is sufficient, the prognosis may be good. Therefore, the Patnaik grade classification alone may be insufficient as a prognostic indicator.
- the present invention provides a method for evaluating the grade of malignancy of a non-human animal tumor, a method for determining a prognosis, and a kit suitable for use of the method.
- the method of the present invention that makes it possible to appropriately determine the prognosis of tumors in non-human animals, particularly pet animals such as dogs and cats, is highly expected to be put to practical use in the field of animal medicine.
Abstract
Provided are a method for evaluating the malignancy of a malignant tumor in a non-human animal and a composition to be used for the evaluation. The method for evaluating the malignancy of a tumor in a non-human animal comprises: detecting CD34 that is expressed in cells of a tumor cell-containing sample derived from the subject animal; and, when the ratio of CD34-expressing cells to the total cells in the sample is at a preset level or higher, evaluating the tumor as malignant. Also provided is a diagnosis kit for evaluating the malignancy of a tumor in a non-human animal, said kit comprising an anti-CD34 antibody.
Description
本発明は、非ヒト動物の悪性腫瘍の悪性度の評価方法に関する。より具体的には、発明の非ヒト動物の悪性腫瘍の悪性度の評価方法には、該腫瘍の予後の予測方法も含まれる。
The present invention relates to a method for evaluating the malignancy of a malignant tumor of a non-human animal. More specifically, the method for evaluating the malignancy of a malignant tumor of a non-human animal of the invention includes a method for predicting the prognosis of the tumor.
肥満細胞腫（Mast cell tumors；MCT）は、イヌやネコに多い腫瘍で、皮膚に発生することが多く、イヌの場合皮膚腫瘍の中で１番多く、ネコでは２番目に多いとされている。
イヌの皮膚肥満細胞腫は、全皮膚腫瘍の7～21%の割合を占める一般的な皮膚腫瘍の1つであり、その生物学的挙動は非常に多様である。臨床的に外科切除で完治する可能性のある良性のものが存在する一方で、領域リンパ節に浸潤するものや外科切除後に再発するもの、致命的な転移を起こすものなど様々なタイプの症例が知られている。そのため、イヌの皮膚肥満細胞腫の悪性度の指標、あるいは、予後を予測するための指標が必要とされてきた。 Mast cell tumors (MCT) are tumors that are common in dogs and cats and often occur in the skin. In dogs, they are the most common skin tumor and the second most common in cats. .
Canine cutaneous mastocytoma is one of the common skin tumors, accounting for 7-21% of all skin tumors, and its biological behavior is very diverse. While there are benign cases that can be completely cured by surgical resection, there are various types of cases such as those that invade regional lymph nodes, those that recur after surgical resection, and those that cause fatal metastases Are known. Therefore, an index of malignancy of canine cutaneous mastocytoma or an index for predicting prognosis has been required.
イヌの皮膚肥満細胞腫は、全皮膚腫瘍の7～21%の割合を占める一般的な皮膚腫瘍の1つであり、その生物学的挙動は非常に多様である。臨床的に外科切除で完治する可能性のある良性のものが存在する一方で、領域リンパ節に浸潤するものや外科切除後に再発するもの、致命的な転移を起こすものなど様々なタイプの症例が知られている。そのため、イヌの皮膚肥満細胞腫の悪性度の指標、あるいは、予後を予測するための指標が必要とされてきた。 Mast cell tumors (MCT) are tumors that are common in dogs and cats and often occur in the skin. In dogs, they are the most common skin tumor and the second most common in cats. .
Canine cutaneous mastocytoma is one of the common skin tumors, accounting for 7-21% of all skin tumors, and its biological behavior is very diverse. While there are benign cases that can be completely cured by surgical resection, there are various types of cases such as those that invade regional lymph nodes, those that recur after surgical resection, and those that cause fatal metastases Are known. Therefore, an index of malignancy of canine cutaneous mastocytoma or an index for predicting prognosis has been required.
皮膚肥満細胞腫の挙動を予測するために臨床ステージ、成長率、腫瘍の存在位置、増殖活性、腫瘍内血管新生などがこれまで指標として研究されてきたが、現在、イヌの皮膚肥満細胞腫の予後及び治療法の決定は主に病理組織学的グレード分類に基づいて行われている。これまで2種類の病理組織学的グレード分類法が提唱されてきたが、イヌの皮膚肥満細胞腫については、1984年にPatnaikらにより提唱されたPatnaikの分類法（非特許文献１）が最も一般的に用いられている。Patnaikの分類法は、病変部の範囲、細胞密度、細胞形態、核***指数、間質反応などに基づいて、高分化型（グレードI）、中等度分化型（グレードII）、低分化型（グレードIII）と分類する。しかしながら、イヌの皮膚肥満細胞腫の大部分はグレードIIに分類されるところ、その予後は様々であり、病理医間の一致率が低いなど、Patnaikの分類法については議論の余地が残っている（非特許文献２）。
従って、イヌの皮膚肥満細胞腫の予後因子を含めた腫瘍の悪性度を評価する因子について病理組織学的分類の他に、近年様々な因子が検討されている。 In order to predict the behavior of cutaneous mastocytoma, clinical stage, growth rate, location of tumor, proliferative activity, intravascular neovascularization, etc. have been studied as indicators. Prognosis and treatment decisions are mainly based on histopathological grade classification. So far, two types of histopathological grade classification methods have been proposed. For dog cutaneous mastocytoma, Patnaik's classification method proposed by Patnaik et al. Has been used. Patnaik's classification method is based on the extent of lesion, cell density, cell morphology, fission index, stromal response, etc., well differentiated (grade I), moderately differentiated (grade II), poorly differentiated (grade) Classify as III). However, the majority of canine cutaneous mastocytomas are classified as grade II, but their prognosis varies, and Patnaik's classification method remains controversial, such as low agreement among pathologists. (Non-patent document 2).
Therefore, in addition to histopathological classification, various factors have been studied in recent years for factors that evaluate tumor malignancy, including prognostic factors for canine cutaneous mastocytoma.
従って、イヌの皮膚肥満細胞腫の予後因子を含めた腫瘍の悪性度を評価する因子について病理組織学的分類の他に、近年様々な因子が検討されている。 In order to predict the behavior of cutaneous mastocytoma, clinical stage, growth rate, location of tumor, proliferative activity, intravascular neovascularization, etc. have been studied as indicators. Prognosis and treatment decisions are mainly based on histopathological grade classification. So far, two types of histopathological grade classification methods have been proposed. For dog cutaneous mastocytoma, Patnaik's classification method proposed by Patnaik et al. Has been used. Patnaik's classification method is based on the extent of lesion, cell density, cell morphology, fission index, stromal response, etc., well differentiated (grade I), moderately differentiated (grade II), poorly differentiated (grade) Classify as III). However, the majority of canine cutaneous mastocytomas are classified as grade II, but their prognosis varies, and Patnaik's classification method remains controversial, such as low agreement among pathologists. (Non-patent document 2).
Therefore, in addition to histopathological classification, various factors have been studied in recent years for factors that evaluate tumor malignancy, including prognostic factors for canine cutaneous mastocytoma.
例えば、予後因子としては、有糸***指数（Mitotic Index；MI）が最も重要な指標の一つとされているが（非特許文献３）、その他に、皮膚肥満細胞腫の腫瘍細胞の増殖度に関与するKi67（MIB-1）核抗原標識指数、核小体形成領域関連蛋白（AgNOR）、増殖性細胞核抗原（PCNA）（非特許文献４～６）や、一部の肥満細胞腫の進行に関与するとされている突然変異したc-kit癌原遺伝子産物であるKITの免疫発現パターン（非特許文献４及び７）、肥満細胞内に存在する中性プロテアーゼの1つであるトリプターゼ（非特許文献７）などが報告されている。
For example, as a prognostic factor, the mitotic index (MI) is one of the most important indices (Non-patent Document 3). In addition, the proliferative factor of tumor cells of cutaneous mastocytoma Involved in the progression of Ki67 (MIB-1) nuclear antigen labeling index, nucleolus formation region-related protein (AgNOR), proliferating cell nuclear antigen (PCNA) (Non-Patent Documents 4-6) and some mastocytomas Immune expression pattern of KIT, a mutated c-kit proto-oncogene product that has been implicated (Non-patent Documents 4 and 7), Tryptase (Non-patent Document), one of the neutral proteases present in mast cells 7) etc. have been reported.
腫瘍の予後を正確に見極めることは、該腫瘍の悪性度を把握する上でも、また、治療方針を決定する上で重要なことであり、とりわけ、外科的手術を行う場合には、切除する範囲を決定する上で必要なことである。イヌの皮膚肥満細胞腫は悪性である場合が少なくなく、外科的手術が必要となる場合が多い中、上記のように、現在のところ、肥満細胞腫の悪性度の評価方法、及び、予後判断の方法は、必ずしも十分な情報を提供しているとは言えない状況にある。
Accurately determining the prognosis of a tumor is important in understanding the malignancy of the tumor and in determining a treatment strategy, and in particular, when performing surgical operations, the extent of resection It is necessary to decide. Canine cutaneous mastocytoma is often malignant and often requires surgical operation. As described above, at present, a method for evaluating the malignancy of mastocytoma and prognosis determination This method does not necessarily provide sufficient information.
上記事情に鑑み、本発明者らは、非ヒト動物の悪性腫瘍の悪性度の評価、及び、予後の予測をより正確に行うための方法を検討した。
すなわち、本発明は、非ヒト動物の悪性腫瘍の悪性度の評価、及び、予後を予測する方法に関する。 In view of the above circumstances, the present inventors examined a method for more accurately evaluating the malignancy of a non-human animal malignant tumor and predicting the prognosis.
That is, the present invention relates to a method for evaluating the malignancy of a malignant tumor of a non-human animal and predicting a prognosis.
すなわち、本発明は、非ヒト動物の悪性腫瘍の悪性度の評価、及び、予後を予測する方法に関する。 In view of the above circumstances, the present inventors examined a method for more accurately evaluating the malignancy of a non-human animal malignant tumor and predicting the prognosis.
That is, the present invention relates to a method for evaluating the malignancy of a malignant tumor of a non-human animal and predicting a prognosis.
発明者らは、イヌの肥満細胞腫の予後あるいは転移の可能性を表す指標として、肥満細胞腫由来の腫瘍細胞におけるCD34の発現状況を手がかりにすることができないか検討を行った。CD34は膜貫通タンパクで、通常、骨格筋の衛星細胞、消化管のカハール介在細胞、血管内皮、脳の神経細胞体、真皮の樹状細胞に発現する。また、CD34は造血幹細胞に発現を認め、CD34陽性細胞は肥満細胞の前駆細胞として知られているため（Kawarai et al., J Vet Med Sci. 2010 Feb;72(2):131-40）、過去に報告されているKITと同様にCD34は皮膚肥満細胞腫の腫瘍細胞の分化度により、その発現に違いが生じる可能性があった。また、CD34を欠損させたマウスより得られた骨髄由来培養肥満細胞は、野生型のマウスから得られた肥満細胞と比べて細胞同士が凝集することも報告されていた（Drew E, et al., Immunity. 2005 Jan;22(1):43-57.）。そこで、発明者らは、新たな予後あるいは転移の因子としての可能性について検討した結果、イヌの皮膚肥満細胞腫由来の試料において、抗CD34抗体による免疫染色のレベルが染色性＋２（後述）以上である細胞の割合が、おおよそ20%以上である場合に、予後が不良となることを初めて見出した。
The inventors examined whether the expression status of CD34 in tumor cells derived from mastocytoma could be used as a clue as an index indicating the prognosis or metastasis potential of canine mastocytoma. CD34 is a transmembrane protein and is usually expressed in skeletal muscle satellite cells, gastrointestinal chahar-mediated cells, vascular endothelium, brain neuronal cell bodies, and dermal dendritic cells. CD34 is also expressed in hematopoietic stem cells, and CD34 positive cells are known as mast cell progenitors (Kawarai et al., J Vet Med Sci. 2010 Feb; 72 (2): 131-40) Like KIT reported in the past, CD34 may have different expression depending on the degree of differentiation of tumor cells of cutaneous mastocytoma. In addition, bone marrow-derived cultured mast cells obtained from CD34-deficient mice have also been reported to aggregate with each other compared to mast cells obtained from wild-type mice (Drew E, et al. , Immunity. 2005 Jan; 22 (1): 43-57.). Therefore, as a result of examining the possibility as a new prognosis or metastasis factor, the inventors have found that the level of immunostaining with an anti-CD34 antibody in a sample derived from canine cutaneous mastocytoma is not less than +2 (described later). It has been found for the first time that the prognosis is poor when the proportion of cells is approximately 20% or more.
すなわち、本発明は以下の（１）～（１１）である。
（１）非ヒト動物の腫瘍の悪性度を評価する方法であって、腫瘍細胞を含む対象動物由来の試料中の細胞に発現しているCD34を検出し、試料中に存在する細胞に対する該CD34を発現する細胞の割合を算出し、その割合に基づいて該腫瘍の悪性度を評価する方法。
（２）前記CD34を発現している細胞の割合が、試料中に存在する細胞の２０％以上である場合に、該腫瘍の悪性度が高いと判断することを特徴とする上記（１）に記載の方法。
（３）前記CD34の発現を検出することが、免疫組織化学的染色法であることを特徴とする上記（１）又は（２）に記載の方法。
（４）前記「CD34を発現する細胞」を、抗CD34抗体による細胞の染色性に基づいて評価することを特徴とする上記（３）に記載の方法。
（５）前記染色性が２＋である場合に、「CD34を発現する細胞」と評価することを特徴とする上記（４）に記載の方法。
（６）前記非ヒト動物が、イヌ又はネコであり、前記腫瘍が皮膚肥満細胞腫であることを特徴とする上記（１）乃至（５）のいずれかに記載の方法。
（７）非ヒト動物の腫瘍の補助療法又は内科的療法の要否を決定するために使用することを特徴とする上記（１）乃至（６）のいずれかに記載の方法。
（８）非ヒト動物の腫瘍の外科手術を行う場合の切除範囲を決定するために使用することを特徴とする上記（１）乃至（６）のいずれかに記載の方法。
（９）非ヒト動物の腫瘍の予後の予測を行うために使用することを特徴とする上記（１）乃至（６）のいずれかに記載の方法。
（１０）抗CD34抗体を含んでなる、非ヒト動物の腫瘍の悪性度を評価するための診断用キット。
（１１）前記非ヒト動物がイヌ又はネコであり、前記腫瘍が皮膚肥満細胞腫であることを特徴とする請求項１０に記載の診断用キット。 That is, the present invention includes the following (1) to (11).
(1) A method for evaluating the malignancy of a tumor in a non-human animal, wherein CD34 expressed in cells in a sample derived from a subject animal containing tumor cells is detected, and the CD34 against the cells present in the sample is detected. The ratio of the cell which expresses is calculated, The method of evaluating the malignancy of this tumor based on the ratio.
(2) The above (1), wherein the tumor is judged to have high malignancy when the ratio of the cells expressing CD34 is 20% or more of the cells present in the sample. The method described.
(3) The method according to (1) or (2) above, wherein the detection of the expression of CD34 is an immunohistochemical staining method.
(4) The method according to (3) above, wherein the “cell expressing CD34” is evaluated on the basis of the staining property of the cell with an anti-CD34 antibody.
(5) The method according to (4) above, wherein when the staining property is 2+, it is evaluated as “a cell expressing CD34”.
(6) The method according to any one of (1) to (5) above, wherein the non-human animal is a dog or a cat, and the tumor is cutaneous mastocytoma.
(7) The method according to any one of (1) to (6) above, wherein the method is used for determining whether or not an adjuvant therapy or a medical therapy for a non-human animal tumor is necessary.
(8) The method according to any one of (1) to (6) above, wherein the method is used to determine a resection range when performing a surgical operation on a tumor of a non-human animal.
(9) The method according to any one of (1) to (6) above, which is used for predicting a prognosis of a tumor of a non-human animal.
(10) A diagnostic kit for evaluating the malignancy of a tumor of a non-human animal, comprising an anti-CD34 antibody.
(11) The diagnostic kit according to claim 10, wherein the non-human animal is a dog or a cat, and the tumor is cutaneous mastocytoma.
（１）非ヒト動物の腫瘍の悪性度を評価する方法であって、腫瘍細胞を含む対象動物由来の試料中の細胞に発現しているCD34を検出し、試料中に存在する細胞に対する該CD34を発現する細胞の割合を算出し、その割合に基づいて該腫瘍の悪性度を評価する方法。
（２）前記CD34を発現している細胞の割合が、試料中に存在する細胞の２０％以上である場合に、該腫瘍の悪性度が高いと判断することを特徴とする上記（１）に記載の方法。
（３）前記CD34の発現を検出することが、免疫組織化学的染色法であることを特徴とする上記（１）又は（２）に記載の方法。
（４）前記「CD34を発現する細胞」を、抗CD34抗体による細胞の染色性に基づいて評価することを特徴とする上記（３）に記載の方法。
（５）前記染色性が２＋である場合に、「CD34を発現する細胞」と評価することを特徴とする上記（４）に記載の方法。
（６）前記非ヒト動物が、イヌ又はネコであり、前記腫瘍が皮膚肥満細胞腫であることを特徴とする上記（１）乃至（５）のいずれかに記載の方法。
（７）非ヒト動物の腫瘍の補助療法又は内科的療法の要否を決定するために使用することを特徴とする上記（１）乃至（６）のいずれかに記載の方法。
（８）非ヒト動物の腫瘍の外科手術を行う場合の切除範囲を決定するために使用することを特徴とする上記（１）乃至（６）のいずれかに記載の方法。
（９）非ヒト動物の腫瘍の予後の予測を行うために使用することを特徴とする上記（１）乃至（６）のいずれかに記載の方法。
（１０）抗CD34抗体を含んでなる、非ヒト動物の腫瘍の悪性度を評価するための診断用キット。
（１１）前記非ヒト動物がイヌ又はネコであり、前記腫瘍が皮膚肥満細胞腫であることを特徴とする請求項１０に記載の診断用キット。 That is, the present invention includes the following (1) to (11).
(1) A method for evaluating the malignancy of a tumor in a non-human animal, wherein CD34 expressed in cells in a sample derived from a subject animal containing tumor cells is detected, and the CD34 against the cells present in the sample is detected. The ratio of the cell which expresses is calculated, The method of evaluating the malignancy of this tumor based on the ratio.
(2) The above (1), wherein the tumor is judged to have high malignancy when the ratio of the cells expressing CD34 is 20% or more of the cells present in the sample. The method described.
(3) The method according to (1) or (2) above, wherein the detection of the expression of CD34 is an immunohistochemical staining method.
(4) The method according to (3) above, wherein the “cell expressing CD34” is evaluated on the basis of the staining property of the cell with an anti-CD34 antibody.
(5) The method according to (4) above, wherein when the staining property is 2+, it is evaluated as “a cell expressing CD34”.
(6) The method according to any one of (1) to (5) above, wherein the non-human animal is a dog or a cat, and the tumor is cutaneous mastocytoma.
(7) The method according to any one of (1) to (6) above, wherein the method is used for determining whether or not an adjuvant therapy or a medical therapy for a non-human animal tumor is necessary.
(8) The method according to any one of (1) to (6) above, wherein the method is used to determine a resection range when performing a surgical operation on a tumor of a non-human animal.
(9) The method according to any one of (1) to (6) above, which is used for predicting a prognosis of a tumor of a non-human animal.
(10) A diagnostic kit for evaluating the malignancy of a tumor of a non-human animal, comprising an anti-CD34 antibody.
(11) The diagnostic kit according to claim 10, wherein the non-human animal is a dog or a cat, and the tumor is cutaneous mastocytoma.
本発明の悪性度の評価方法及び予後の予測方法によれば、非ヒト動物の腫瘍細胞、特に肥満細胞腫の悪性度及び予後を的確に診断することが可能である。
According to the malignancy evaluation method and prognosis prediction method of the present invention, it is possible to accurately diagnose the malignancy and prognosis of tumor cells of non-human animals, particularly mastocytoma.
また、本発明の悪性度の評価方法及び予後の予測方法は、腫瘍の切除範囲を決定するための手がかりを提供することができる。
Further, the malignancy evaluation method and prognosis prediction method of the present invention can provide a clue for determining the resection range of a tumor.
これまでは、外科手術を行わない限り、悪性度の判定、あるいは、予後判定をすることができず、全ての症例において、化学療法の適応を考える必要性があった。
本発明により、転移の可能性について、画像診断を行わなくとも予測が可能となり、化学療法の開始を本発明の結果に沿って行うことができる。すなわち、必要な症例に適切な化学療法を行うことができる。この点、本発明は、陰性である症例には、不必要な化学療法をしなくてよいという効果も発揮する。 Until now, unless surgery was performed, malignancy or prognosis could not be determined, and it was necessary to consider the indication of chemotherapy in all cases.
According to the present invention, the possibility of metastasis can be predicted without performing image diagnosis, and chemotherapy can be started in accordance with the results of the present invention. That is, it is possible to perform appropriate chemotherapy for necessary cases. In this respect, the present invention also exerts an effect that it is not necessary to perform unnecessary chemotherapy for a negative case.
本発明により、転移の可能性について、画像診断を行わなくとも予測が可能となり、化学療法の開始を本発明の結果に沿って行うことができる。すなわち、必要な症例に適切な化学療法を行うことができる。この点、本発明は、陰性である症例には、不必要な化学療法をしなくてよいという効果も発揮する。 Until now, unless surgery was performed, malignancy or prognosis could not be determined, and it was necessary to consider the indication of chemotherapy in all cases.
According to the present invention, the possibility of metastasis can be predicted without performing image diagnosis, and chemotherapy can be started in accordance with the results of the present invention. That is, it is possible to perform appropriate chemotherapy for necessary cases. In this respect, the present invention also exerts an effect that it is not necessary to perform unnecessary chemotherapy for a negative case.
本発明は、腫瘍細胞に発現するCD34の発現量を指標にして、該腫瘍細胞の悪性度の評価、予後若しくは転移の有無を予測する方法である。
すなわち、本発明の実施形態の１つは、非ヒト動物の腫瘍の悪性度の評価、あるいは、予後の予測方法であって、腫瘍細胞を含む対象動物由来の試料中の細胞に発現しているCD34の発現を検出し、該CD34を発現する細胞数が、試料中に存在する全細胞数の一定割合以上の場合に、該腫瘍の悪性度が高い、又は、予後が不良であると判断する方法である。
ここで、「悪性度が高い」とは、その後の予後が悪いことを意味し、本発明の腫瘍の悪性度の評価方法を使用して、得られた悪性度は、該腫瘍の外科的手術による切除範囲の決定、術前又は術後の補助療法若しくは内科的療法（化学療法剤や、分子標的薬を使用した療法など）の要否などの治療方法の選択を行う上で有効である。従って、該腫瘍の治療範囲や治療方法を決定するために、本発明を使用することは、当然に、本発明の権利範囲内における本発明の実施に該当する。
また、「予後」とは、医学分野で用いられる場合の意味と同じであって、特に限定はしないが、例えば、予想される医学的な状態（健康状態）に関する見解、病気・創傷の将来的な状態のことである。また、疾患が癌等である場合に、予後の評価又は予後の診断としては、例えば、癌等のグレード診断、悪性度診断、将来における転移の有無の予測、手術後の症状の悪化の有無の予測等を挙げることができる。そして、予後が不良であるとは、例えば、生存率の短縮、再発リスクの増大及び／又は腫瘍が他の部位に転移している可能性がある場合を指す。また、本発明における腫瘍細胞は、CD34を発現する細胞を起源とする細胞、例えば、毛包幹細胞、肥満細胞、血管内皮細胞、線維芽細胞、ランゲルハンス細胞を起源とする細胞が腫瘍化したものであり、特に好ましくは、肥満細胞腫細胞である。本発明の予後評価方法の対象となる非ヒト動物は、特に限定はしないが、例えば、イヌ、ネコ、フェレットなどである。 The present invention is a method for evaluating the malignancy of a tumor cell, predicting the prognosis, or the presence of metastasis using the expression level of CD34 expressed in the tumor cell as an index.
That is, one of the embodiments of the present invention is a method for evaluating the malignancy of a tumor in a non-human animal or predicting prognosis, and is expressed in cells in a sample derived from a target animal including tumor cells. When the expression of CD34 is detected and the number of cells expressing CD34 is a certain percentage or more of the total number of cells present in the sample, it is judged that the tumor has a high malignancy or a poor prognosis Is the method.
Here, “high malignancy” means that the prognosis thereafter is poor, and the malignancy obtained using the tumor malignancy evaluation method of the present invention is determined by surgical operation of the tumor. This is effective in determining the range of resection, and the choice of treatment methods such as the necessity of pre- or post-operative adjuvant therapy or medical therapy (such as chemotherapy using chemotherapeutic agents or molecular targeted drugs). Accordingly, the use of the present invention to determine the therapeutic range and method of treatment of the tumor naturally falls within the scope of the present invention within the scope of the present invention.
In addition, “prognosis” has the same meaning as used in the medical field and is not particularly limited. For example, a view regarding an expected medical condition (health condition), a future of illness / wound It is a state. In addition, when the disease is cancer or the like, the prognostic evaluation or prognosis includes, for example, grade diagnosis of cancer, malignancy diagnosis, prediction of the presence or absence of metastasis in the future, presence or absence of deterioration of symptoms after surgery Predictions can be mentioned. The poor prognosis refers to, for example, a case where there is a possibility that the survival rate is shortened, the risk of recurrence is increased, and / or the tumor has metastasized to another site. In addition, the tumor cells in the present invention are cells that originate from cells expressing CD34, such as cells derived from hair follicle stem cells, mast cells, vascular endothelial cells, fibroblasts, and Langerhans cells. Particularly preferred are mastocytoma cells. The non-human animal that is the subject of the prognostic evaluation method of the present invention is not particularly limited, and examples thereof include dogs, cats, ferrets, and the like.
すなわち、本発明の実施形態の１つは、非ヒト動物の腫瘍の悪性度の評価、あるいは、予後の予測方法であって、腫瘍細胞を含む対象動物由来の試料中の細胞に発現しているCD34の発現を検出し、該CD34を発現する細胞数が、試料中に存在する全細胞数の一定割合以上の場合に、該腫瘍の悪性度が高い、又は、予後が不良であると判断する方法である。
ここで、「悪性度が高い」とは、その後の予後が悪いことを意味し、本発明の腫瘍の悪性度の評価方法を使用して、得られた悪性度は、該腫瘍の外科的手術による切除範囲の決定、術前又は術後の補助療法若しくは内科的療法（化学療法剤や、分子標的薬を使用した療法など）の要否などの治療方法の選択を行う上で有効である。従って、該腫瘍の治療範囲や治療方法を決定するために、本発明を使用することは、当然に、本発明の権利範囲内における本発明の実施に該当する。
また、「予後」とは、医学分野で用いられる場合の意味と同じであって、特に限定はしないが、例えば、予想される医学的な状態（健康状態）に関する見解、病気・創傷の将来的な状態のことである。また、疾患が癌等である場合に、予後の評価又は予後の診断としては、例えば、癌等のグレード診断、悪性度診断、将来における転移の有無の予測、手術後の症状の悪化の有無の予測等を挙げることができる。そして、予後が不良であるとは、例えば、生存率の短縮、再発リスクの増大及び／又は腫瘍が他の部位に転移している可能性がある場合を指す。また、本発明における腫瘍細胞は、CD34を発現する細胞を起源とする細胞、例えば、毛包幹細胞、肥満細胞、血管内皮細胞、線維芽細胞、ランゲルハンス細胞を起源とする細胞が腫瘍化したものであり、特に好ましくは、肥満細胞腫細胞である。本発明の予後評価方法の対象となる非ヒト動物は、特に限定はしないが、例えば、イヌ、ネコ、フェレットなどである。 The present invention is a method for evaluating the malignancy of a tumor cell, predicting the prognosis, or the presence of metastasis using the expression level of CD34 expressed in the tumor cell as an index.
That is, one of the embodiments of the present invention is a method for evaluating the malignancy of a tumor in a non-human animal or predicting prognosis, and is expressed in cells in a sample derived from a target animal including tumor cells. When the expression of CD34 is detected and the number of cells expressing CD34 is a certain percentage or more of the total number of cells present in the sample, it is judged that the tumor has a high malignancy or a poor prognosis Is the method.
Here, “high malignancy” means that the prognosis thereafter is poor, and the malignancy obtained using the tumor malignancy evaluation method of the present invention is determined by surgical operation of the tumor. This is effective in determining the range of resection, and the choice of treatment methods such as the necessity of pre- or post-operative adjuvant therapy or medical therapy (such as chemotherapy using chemotherapeutic agents or molecular targeted drugs). Accordingly, the use of the present invention to determine the therapeutic range and method of treatment of the tumor naturally falls within the scope of the present invention within the scope of the present invention.
In addition, “prognosis” has the same meaning as used in the medical field and is not particularly limited. For example, a view regarding an expected medical condition (health condition), a future of illness / wound It is a state. In addition, when the disease is cancer or the like, the prognostic evaluation or prognosis includes, for example, grade diagnosis of cancer, malignancy diagnosis, prediction of the presence or absence of metastasis in the future, presence or absence of deterioration of symptoms after surgery Predictions can be mentioned. The poor prognosis refers to, for example, a case where there is a possibility that the survival rate is shortened, the risk of recurrence is increased, and / or the tumor has metastasized to another site. In addition, the tumor cells in the present invention are cells that originate from cells expressing CD34, such as cells derived from hair follicle stem cells, mast cells, vascular endothelial cells, fibroblasts, and Langerhans cells. Particularly preferred are mastocytoma cells. The non-human animal that is the subject of the prognostic evaluation method of the present invention is not particularly limited, and examples thereof include dogs, cats, ferrets, and the like.
CD34は、110kDaの単鎖膜貫通型リン酸化糖タンパク質のことであるが、本明細書において「CD34」と記載する場合は、タンパク質のことを指し、これをコードする核酸等については、「CD34核酸」と記載する。CD34のアミノ酸配列及び核酸配列等の情報はすでに公開のデータベース等に開示されているため、当業者であれば、容易に取得することが可能である。参考まで挙げるとすれば、イヌのCD34アミノ酸配列及び核酸配列は、各々、配列番号１及び２であり、ネコのCD34アミノ酸配列及び核酸配列は、各々配列番号３及び４である。
CD34 is a 110 kDa single-chain transmembrane phosphorylated glycoprotein, but when described herein as “CD34”, it refers to a protein. "Nucleic acid". Information such as the amino acid sequence and nucleic acid sequence of CD34 has already been disclosed in public databases, and can be easily obtained by those skilled in the art. For reference, the canine CD34 amino acid sequence and nucleic acid sequence are SEQ ID NOs: 1 and 2, respectively, and the feline CD34 amino acid sequence and nucleic acid sequence are SEQ ID NOs: 3 and 4, respectively.
本発明の好ましい実施形態には、CD34を検出する工程が含まれる。この場合、診断対象動物からの腫瘍細胞の試料を採取する手段は、当業者において、容易に選択されるいかなる方法、例えば、穿刺針を用いて細胞試料を得る針生検、外科的に切開して患部組織片を得る切開生検などの方法により実施することができる。
A preferred embodiment of the present invention includes the step of detecting CD34. In this case, means for collecting a sample of tumor cells from the animal to be diagnosed may be any method easily selected by those skilled in the art, for example, a needle biopsy for obtaining a cell sample using a puncture needle, or a surgical incision. It can be carried out by a method such as an incision biopsy to obtain an affected tissue piece.
腫瘍細胞を含む診断試料中におけるCD34の発現状況については、当業者において容易に選択し得る方法により実施することができる。
例えば、免疫組織化学的手法により、試料中の腫瘍細胞に発現するCD34を検出し、その発現レベルを調べる場合、適当な組織標本あるいは細胞診標本を作製し、検討を行うことができる。組織標本あるいは細胞標本の作製方法は、既知のいかなる方法を使用して作製してもよい。例えば、採取した組織等をホルマリン等で固定し、パラフィン包埋処理後、切片を作製し、免疫組織化学染色を行いCD34の発現レベルの検討を行うことができる。あるいは、細胞診の１つの方法である、Liquid Based Cytology（LBC: 液状化細胞診）により実施することもできる。LBC法は、採取した細胞診検体（腫瘍細胞試料）を分散液（保存液）中で撹拌・分散した後、細胞を回収しスライドガラス上へ薄く転写・塗沫し、固定した後、抗体染色等を行い染色されるCD34の量を調べる方法である。また、スライドガラス標本ではなくとも、細胞を分散した状態で試料中のCD34を発現する細胞を検出するフローサイトメトリー法を用いてもよい。 The expression state of CD34 in a diagnostic sample containing tumor cells can be carried out by a method that can be easily selected by those skilled in the art.
For example, when CD34 expressed in tumor cells in a sample is detected by immunohistochemical technique and the expression level is examined, an appropriate tissue specimen or cytological specimen can be prepared and examined. The tissue specimen or cell specimen may be produced using any known method. For example, the collected tissue or the like can be fixed with formalin or the like, and after paraffin embedding treatment, a section can be prepared and immunohistochemical staining can be performed to examine the expression level of CD34. Or it can also implement by Liquid Based Cytology (LBC: Liquefaction cytology) which is one method of cytodiagnosis. In the LBC method, collected cytological specimens (tumor cell samples) are stirred and dispersed in a dispersion (preservation solution), then the cells are collected, transferred and smeared onto a glass slide, fixed, and then stained with antibodies. This is a method for examining the amount of CD34 stained. Further, a flow cytometry method for detecting cells expressing CD34 in a sample in a dispersed state may be used instead of the slide glass specimen.
例えば、免疫組織化学的手法により、試料中の腫瘍細胞に発現するCD34を検出し、その発現レベルを調べる場合、適当な組織標本あるいは細胞診標本を作製し、検討を行うことができる。組織標本あるいは細胞標本の作製方法は、既知のいかなる方法を使用して作製してもよい。例えば、採取した組織等をホルマリン等で固定し、パラフィン包埋処理後、切片を作製し、免疫組織化学染色を行いCD34の発現レベルの検討を行うことができる。あるいは、細胞診の１つの方法である、Liquid Based Cytology（LBC: 液状化細胞診）により実施することもできる。LBC法は、採取した細胞診検体（腫瘍細胞試料）を分散液（保存液）中で撹拌・分散した後、細胞を回収しスライドガラス上へ薄く転写・塗沫し、固定した後、抗体染色等を行い染色されるCD34の量を調べる方法である。また、スライドガラス標本ではなくとも、細胞を分散した状態で試料中のCD34を発現する細胞を検出するフローサイトメトリー法を用いてもよい。 The expression state of CD34 in a diagnostic sample containing tumor cells can be carried out by a method that can be easily selected by those skilled in the art.
For example, when CD34 expressed in tumor cells in a sample is detected by immunohistochemical technique and the expression level is examined, an appropriate tissue specimen or cytological specimen can be prepared and examined. The tissue specimen or cell specimen may be produced using any known method. For example, the collected tissue or the like can be fixed with formalin or the like, and after paraffin embedding treatment, a section can be prepared and immunohistochemical staining can be performed to examine the expression level of CD34. Or it can also implement by Liquid Based Cytology (LBC: Liquefaction cytology) which is one method of cytodiagnosis. In the LBC method, collected cytological specimens (tumor cell samples) are stirred and dispersed in a dispersion (preservation solution), then the cells are collected, transferred and smeared onto a glass slide, fixed, and then stained with antibodies. This is a method for examining the amount of CD34 stained. Further, a flow cytometry method for detecting cells expressing CD34 in a sample in a dispersed state may be used instead of the slide glass specimen.
採取した組織又は細胞中に発現しているCD34を免疫組織化学的手法によって検出するため、CD34に対する抗体（抗CD34抗体）を使用することができる。抗CD34抗体は、本発明を実施する者がみずから作製した抗体、市販されている抗体（例えば、SANTA CRUZ BIOTECHNOLOGY社、#sc-7045）のいずれであっても使用可能であり、また、モノクローナル抗体でもポリクローナル抗体でもよい。さらに、完全体の抗体である必要はなく、CDR領域等を含む断片であっても良く、遺伝子工学的に調製されたものであっても良い。抗体の断片としては、細胞上に発現しているCD34と結合し、免疫組織化学的染色に用いることができるものであれば如何なるものであってもよく、例えば、抗CD34抗体の一部分の領域を含むペプチド断片である、Fab、Fab’、F(ab’)2、Fv(variablefragment of antibody)、一本鎖抗体（重鎖、軽鎖、重鎖可変領域、及び軽鎖可変領域等）、scFv、diabody(scFv二量体)、dsFv（ジスルフィド安定化可変領域）、並びに、CDRを少なくとも一部に含むペプチド等が挙げられる。
An antibody against CD34 (anti-CD34 antibody) can be used to detect CD34 expressed in collected tissues or cells by an immunohistochemical technique. As the anti-CD34 antibody, any one of an antibody prepared by a person who carries out the present invention or a commercially available antibody (for example, SANTARUCRUZ BIOTECHNOLOGY, # sc-7045) can be used. Or a polyclonal antibody may be sufficient. Further, the antibody does not have to be a complete antibody, and may be a fragment containing a CDR region or the like, or may be prepared by genetic engineering. The antibody fragment may be any fragment that binds to CD34 expressed on cells and can be used for immunohistochemical staining. For example, a partial region of an anti-CD34 antibody Fab, Fab ′, F (ab ′) 2, Fv (variablefragment of antibody), single chain antibody (heavy chain, light chain, heavy chain variable region, light chain variable region, etc.), scFv , Diabody (scFv dimer), dsFv (disulfide stabilization variable region), and a peptide containing CDR at least in part.
診断対象動物から取得した組織又は細胞試料を、抗CD34抗体を用いて免疫染色する場合、抗CD34抗体、あるいは、抗CD34抗体を１次抗体として使用する場合には該抗CD34抗体と結合する２次抗体に適当な標識を結合させて、その標識を視覚化することで実施することができる。例えば、ペルオキシダーゼで標識した場合には、ジアミノベンチジン（DAB）又はアミノメチルカルバゾール（ACE）などを発色基質とし、アルカリフォスファターゼで標識した場合には、5-ブロモ-4-クロロ-3-インドキシルフォスフェート／ニトロブルーテトラゾリウムクロライド（BCIP/NBT）などを発色基質として使用し、染色することができる。
次に、免疫組織化学的方法により、細胞におけるCD34発現状況を評価する場合、例えば、Jimenez et al., Mod Pathol., 13:37-45, 2000に記載の方法に従って行うことができる。すなわち、低倍率で染色した組織又は細胞標本を顕微鏡観察し、最も染色強度が強い領域を選択し、次いで、その領域を高倍率視野下で観察を行い、観察対象の細胞１００個を選ぶ。
選んだ１００個の細胞の各々の染色強度を以下の４つに分類する（図１も参照のこと）。
０：染色性が認められない、あるいは、バックグラウンドと同程度にわずかに染色されている細胞。
１＋：低倍率では０と区別がつかないが、高倍率で淡く染色性が確認される細胞。
２＋：低倍率で染色性が確認可能で、高倍率で完全に染色性が確認される細胞。
３＋：低倍率で完全に染色性が確認される細胞。
選んだ１００個の細胞に対する染色性２＋以上の細胞を、「CD34を発現している細胞」と判定しその割合を算出し、その割合が１０％以上、１５％以上、より好ましくは２０％以上の場合に、抗CD34抗体による染色性が「陽性」であると判断する。そして、「陽性」と判断された試料が由来する腫瘍細胞の予後は、不良であると評価することができる。
また、採取された試料の染色性の状態をカメラ等で撮影し、染色処理した細胞の画像を取得し、当該画像を電子情報化処理し、解析することも可能である。画像から得られる染色強度を定量し、数値化することで、上記の４段階の染色レベルを評価してもよい。例えば、顕微鏡上で主となる腫瘍細胞の集団を染色した強倍率視野像（200から400倍）を撮影し、撮影した画像内のCD34発現陽性と染色された色を、バイオイメージング解析システム（Lumina Vision, 三谷商事）を用いて、赤、緑、青のRGBカラーに分け、色の染色性を各々、色相によって表して、二値化する。撮影した画像上の染色された陽性面積を、陰性抗体によって非特異的に染色された陰性面積を引くことにより求め、陽性面積と転移および生存結果の間の統計解析からROC曲線を求めてカットオフ値を設定し、カットオフ値以上を陽性と判定することができる。 When immunostaining a tissue or cell sample obtained from an animal to be diagnosed with an anti-CD34 antibody, it binds to the anti-CD34 antibody when an anti-CD34 antibody or an anti-CD34 antibody is used as a primary antibody 2 This can be done by attaching an appropriate label to the next antibody and visualizing the label. For example, when labeled with peroxidase, diaminobenzidine (DAB) or aminomethylcarbazole (ACE) is used as a chromogenic substrate, and when labeled with alkaline phosphatase, 5-bromo-4-chloro-3-indoxyl Phosphate / nitro blue tetrazolium chloride (BCIP / NBT) can be used as a chromogenic substrate for staining.
Next, when the CD34 expression status in a cell is evaluated by an immunohistochemical method, it can be performed according to the method described in Jimenez et al., Mod Pathol., 13: 37-45, 2000, for example. That is, a tissue or cell specimen stained at a low magnification is observed with a microscope, a region having the strongest staining intensity is selected, and then the region is observed under a high-magnification visual field to select 100 cells to be observed.
The staining intensity of each of the selected 100 cells is classified into the following four (see also FIG. 1).
0: Cells that are not stained or are slightly stained as much as the background.
1+: Cells that are indistinguishable from 0 at a low magnification, but are light and stainable at a high magnification.
2+: Cells whose staining property can be confirmed at a low magnification and completely stained at a high magnification.
3+: Cells that are completely stained at low magnification.
Cells having a staining ability of 2+ or more with respect to 100 selected cells are determined as “cells expressing CD34”, and the ratio is calculated. The ratio is 10% or more, 15% or more, more preferably 20% or more. In this case, it is determined that the staining property with the anti-CD34 antibody is “positive”. And it can be evaluated that the prognosis of the tumor cell from which the sample judged to be "positive" is derived is poor.
It is also possible to photograph the stained state of the collected sample with a camera or the like, obtain an image of the stained cell, perform electronic information processing on the image, and analyze the image. The above four levels of staining may be evaluated by quantifying and digitizing the staining intensity obtained from the image. For example, a high-magnification field image (200 to 400 times) of a main tumor cell group stained on a microscope is photographed, and the color stained with CD34 expression positive in the photographed image is displayed as a bioimaging analysis system (Lumina Vision, Mitani Corp.) and divide it into red, green, and blue RGB colors. The stained positive area on the photographed image is obtained by subtracting the negative area that is non-specifically stained with the negative antibody, and the ROC curve is obtained by statistical analysis between the positive area and the metastasis and survival results to cut off. A value can be set and a cutoff value or higher can be determined as positive.
次に、免疫組織化学的方法により、細胞におけるCD34発現状況を評価する場合、例えば、Jimenez et al., Mod Pathol., 13:37-45, 2000に記載の方法に従って行うことができる。すなわち、低倍率で染色した組織又は細胞標本を顕微鏡観察し、最も染色強度が強い領域を選択し、次いで、その領域を高倍率視野下で観察を行い、観察対象の細胞１００個を選ぶ。
選んだ１００個の細胞の各々の染色強度を以下の４つに分類する（図１も参照のこと）。
０：染色性が認められない、あるいは、バックグラウンドと同程度にわずかに染色されている細胞。
１＋：低倍率では０と区別がつかないが、高倍率で淡く染色性が確認される細胞。
２＋：低倍率で染色性が確認可能で、高倍率で完全に染色性が確認される細胞。
３＋：低倍率で完全に染色性が確認される細胞。
選んだ１００個の細胞に対する染色性２＋以上の細胞を、「CD34を発現している細胞」と判定しその割合を算出し、その割合が１０％以上、１５％以上、より好ましくは２０％以上の場合に、抗CD34抗体による染色性が「陽性」であると判断する。そして、「陽性」と判断された試料が由来する腫瘍細胞の予後は、不良であると評価することができる。
また、採取された試料の染色性の状態をカメラ等で撮影し、染色処理した細胞の画像を取得し、当該画像を電子情報化処理し、解析することも可能である。画像から得られる染色強度を定量し、数値化することで、上記の４段階の染色レベルを評価してもよい。例えば、顕微鏡上で主となる腫瘍細胞の集団を染色した強倍率視野像（200から400倍）を撮影し、撮影した画像内のCD34発現陽性と染色された色を、バイオイメージング解析システム（Lumina Vision, 三谷商事）を用いて、赤、緑、青のRGBカラーに分け、色の染色性を各々、色相によって表して、二値化する。撮影した画像上の染色された陽性面積を、陰性抗体によって非特異的に染色された陰性面積を引くことにより求め、陽性面積と転移および生存結果の間の統計解析からROC曲線を求めてカットオフ値を設定し、カットオフ値以上を陽性と判定することができる。 When immunostaining a tissue or cell sample obtained from an animal to be diagnosed with an anti-CD34 antibody, it binds to the anti-CD34 antibody when an anti-CD34 antibody or an anti-CD34 antibody is used as a primary antibody 2 This can be done by attaching an appropriate label to the next antibody and visualizing the label. For example, when labeled with peroxidase, diaminobenzidine (DAB) or aminomethylcarbazole (ACE) is used as a chromogenic substrate, and when labeled with alkaline phosphatase, 5-bromo-4-chloro-3-indoxyl Phosphate / nitro blue tetrazolium chloride (BCIP / NBT) can be used as a chromogenic substrate for staining.
Next, when the CD34 expression status in a cell is evaluated by an immunohistochemical method, it can be performed according to the method described in Jimenez et al., Mod Pathol., 13: 37-45, 2000, for example. That is, a tissue or cell specimen stained at a low magnification is observed with a microscope, a region having the strongest staining intensity is selected, and then the region is observed under a high-magnification visual field to select 100 cells to be observed.
The staining intensity of each of the selected 100 cells is classified into the following four (see also FIG. 1).
0: Cells that are not stained or are slightly stained as much as the background.
1+: Cells that are indistinguishable from 0 at a low magnification, but are light and stainable at a high magnification.
2+: Cells whose staining property can be confirmed at a low magnification and completely stained at a high magnification.
3+: Cells that are completely stained at low magnification.
Cells having a staining ability of 2+ or more with respect to 100 selected cells are determined as “cells expressing CD34”, and the ratio is calculated. The ratio is 10% or more, 15% or more, more preferably 20% or more. In this case, it is determined that the staining property with the anti-CD34 antibody is “positive”. And it can be evaluated that the prognosis of the tumor cell from which the sample judged to be "positive" is derived is poor.
It is also possible to photograph the stained state of the collected sample with a camera or the like, obtain an image of the stained cell, perform electronic information processing on the image, and analyze the image. The above four levels of staining may be evaluated by quantifying and digitizing the staining intensity obtained from the image. For example, a high-magnification field image (200 to 400 times) of a main tumor cell group stained on a microscope is photographed, and the color stained with CD34 expression positive in the photographed image is displayed as a bioimaging analysis system (Lumina Vision, Mitani Corp.) and divide it into red, green, and blue RGB colors. The stained positive area on the photographed image is obtained by subtracting the negative area that is non-specifically stained with the negative antibody, and the ROC curve is obtained by statistical analysis between the positive area and the metastasis and survival results to cut off. A value can be set and a cutoff value or higher can be determined as positive.
また、いわゆるハイブリダイゼーション法により、試料中の腫瘍細胞に発現するCD34のmRNAを検出し、CD34の発現状況をモニターしてもよい。使用可能なハイブリダイゼーション法として、例えば、in situ ハイブリダイゼーション法などを挙げることができる（例えば、Pascucci et al., Vet Dermatol. 2006 Aug;17(4):244-51などを参照のこと）。予後診断の対象となる腫瘍から取得した組織切片又は細胞標本に対し、CD34のmRNAに相補的な標識プローブ、例えば、放射性標識プローブ、ジゴキシゲニン（DIG）プローブ、蛍光標識（FITC, RITCなど）プローブなどを使用して、試料切片又は標本中におけるCD34のmRNA量を検出することができる。試料中のCD34のmRNA量の評価は、標識プローブから得られるシグナルを顕微鏡下で観察し、標識からシグナル強度に基づいて、観察される細胞を上記〔００１９〕のように４段階に分類し、シグナル強度が２＋以上の細胞の割合を算出して、予後の判断の指標とすることができる。
その他、免疫組織化学的方法以外にも、試料中の腫瘍細胞に発現するCD34のレベルを検出する方法として、定量RT-PCR（real time PCR）法により、試料中のCD34 mRNAの発現量を検出してもよい。 Alternatively, CD34 mRNA expressed in tumor cells in a sample may be detected by a so-called hybridization method to monitor the expression status of CD34. Examples of hybridization methods that can be used include in situ hybridization methods (see, for example, Pascucci et al., Vet Dermatol. 2006 Aug; 17 (4): 244-51). Labeled probes complementary to CD34 mRNA, such as radioactively labeled probes, digoxigenin (DIG) probes, fluorescently labeled (FITC, RITC, etc.) probes, etc. Can be used to detect the amount of CD34 mRNA in a sample section or specimen. Evaluation of the amount of mRNA of CD34 in the sample is performed by observing the signal obtained from the labeled probe under a microscope, classifying the observed cells into four stages as described above [0019] based on the signal intensity from the label, The ratio of cells having a signal intensity of 2+ or more can be calculated and used as an indicator of prognosis.
In addition to immunohistochemical methods, the amount of CD34 mRNA expressed in a sample is detected by quantitative RT-PCR (real time PCR) as a method for detecting the level of CD34 expressed in tumor cells in the sample. May be.
その他、免疫組織化学的方法以外にも、試料中の腫瘍細胞に発現するCD34のレベルを検出する方法として、定量RT-PCR（real time PCR）法により、試料中のCD34 mRNAの発現量を検出してもよい。 Alternatively, CD34 mRNA expressed in tumor cells in a sample may be detected by a so-called hybridization method to monitor the expression status of CD34. Examples of hybridization methods that can be used include in situ hybridization methods (see, for example, Pascucci et al., Vet Dermatol. 2006 Aug; 17 (4): 244-51). Labeled probes complementary to CD34 mRNA, such as radioactively labeled probes, digoxigenin (DIG) probes, fluorescently labeled (FITC, RITC, etc.) probes, etc. Can be used to detect the amount of CD34 mRNA in a sample section or specimen. Evaluation of the amount of mRNA of CD34 in the sample is performed by observing the signal obtained from the labeled probe under a microscope, classifying the observed cells into four stages as described above [0019] based on the signal intensity from the label, The ratio of cells having a signal intensity of 2+ or more can be calculated and used as an indicator of prognosis.
In addition to immunohistochemical methods, the amount of CD34 mRNA expressed in a sample is detected by quantitative RT-PCR (real time PCR) as a method for detecting the level of CD34 expressed in tumor cells in the sample. May be.
本発明の他の実施形態は、イヌ又はネコ等の非ヒト動物に発症する腫瘍細胞、例えば、肥満細胞腫の悪性度の評価用キット、あるいは、予後の診断用キットである。上述のように、本発明は、CD34の腫瘍細胞における発現レベルを指標にして、該腫瘍細胞の悪性度の評価、及び、予後を予測する方法を提供するものである。従って、試料中に含まれる腫瘍細胞におけるCD34の発現量を測定するために使用する、抗CD34抗体やCD34 mRNAの発現量を測定するために使用するプローブ、又はプライマー等は、非ヒト動物の予後を診断するための用途を有しており、該用途は本発明において初めて開示されるものである。本発明の腫瘍細胞の予後の診断用キットには、その必須の構成物として細胞に発現しているCD34を検出するためのもの、例えば、抗CD34抗体、CD34 mRNAを検出するためのプローブが含まれる。それ以外に、付属的な構成物として、例えば、診断対象となる組織又は細胞を免疫染色するために必要な、ホルマリン等の固定剤の他、免疫組織化学染色、あるいは、定量RT-PCRを行うために必要な発色基質やバッファー等を含んでいてもよい。
Another embodiment of the present invention is a kit for evaluating malignancy of a tumor cell that develops in a non-human animal such as a dog or a cat, for example, a mastocytoma, or a prognosis diagnostic kit. As described above, the present invention provides a method for evaluating the malignancy of a tumor cell and predicting the prognosis using the expression level of CD34 in the tumor cell as an index. Therefore, the probe or primer used to measure the expression level of anti-CD34 antibody or CD34 mRNA used to measure the expression level of CD34 in tumor cells contained in the sample is the prognosis of non-human animals. This application is disclosed for the first time in the present invention. The diagnostic kit for prognosis of tumor cells of the present invention includes a probe for detecting CD34 expressed in cells as an essential component thereof, for example, an anti-CD34 antibody, a probe for detecting CD34 mRNA. It is. In addition, as an accessory component, for example, in addition to a fixing agent such as formalin necessary for immunostaining a tissue or cells to be diagnosed, immunohistochemical staining or quantitative RT-PCR is performed. For this purpose, it may contain a chromogenic substrate, a buffer and the like.
以下に実施例を示してさらに詳細に説明するが、本発明は以下の実施例により何ら限定されるものではない。
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
１．実験方法
１－１．供試動物
2007年から2011年の間に麻布大学附属動物病院腫瘍科に来院し、外科切除術を受けて腫瘤を摘出したのち、同病院病理検査室に提出されて肥満細胞腫と診断された15症例を対象とした。それぞれの症例についての臨床経過、採材年月日、年齢、性別、犬種、体重、腫瘤病変の分布と数、腫瘤の発生部位、外科的マージンを含む情報は、病理組織検査依頼書から得られた。症例に関する情報を表1にまとめた。 1. Experimental method 1-1. Test animals Visited the department of oncology at Azabu University Hospital between 2007 and 2011. After surgical removal, the tumor was removed and submitted to the pathology laboratory of the hospital to be diagnosed with mastocytoma. Fifteen cases were included. Information about the clinical course, date of collection, age, sex, dog breed, body weight, distribution and number of mass lesions, location of mass, and surgical margin for each case can be obtained from the histopathology request form. It was. Information about the cases is summarized in Table 1.
１－１．供試動物
2007年から2011年の間に麻布大学附属動物病院腫瘍科に来院し、外科切除術を受けて腫瘤を摘出したのち、同病院病理検査室に提出されて肥満細胞腫と診断された15症例を対象とした。それぞれの症例についての臨床経過、採材年月日、年齢、性別、犬種、体重、腫瘤病変の分布と数、腫瘤の発生部位、外科的マージンを含む情報は、病理組織検査依頼書から得られた。症例に関する情報を表1にまとめた。 1. Experimental method 1-1. Test animals Visited the department of oncology at Azabu University Hospital between 2007 and 2011. After surgical removal, the tumor was removed and submitted to the pathology laboratory of the hospital to be diagnosed with mastocytoma. Fifteen cases were included. Information about the clinical course, date of collection, age, sex, dog breed, body weight, distribution and number of mass lesions, location of mass, and surgical margin for each case can be obtained from the histopathology request form. It was. Information about the cases is summarized in Table 1.
１－２． 免疫染色標本の作成手順
（１）固定と切り出し
組織検体は外科切除後直ちに10％中性緩衝ホルマリンに浸漬し、室温下で18～23時間の一次固定を行った。単発性小型の腫瘤は周囲組織までを含むように、単発性大型の腫瘤は分割し、多発性腫瘤は、それぞれ個々の大きさに応じて行った。症例1～5、7、10～12は単発性小型腫瘤であった。症例6、13は多発性腫瘤であった。症例8、9、14、15は単発性大型腫瘤で、それぞれ症例8からは3ブロック、症例9からは4ブロック、症例14からは3ブロック、症例15からは1ブロック作成した。 1-2. Preparation procedure of immunostained specimen (1) Fixation and excision Tissue specimens were immersed in 10% neutral buffered formalin immediately after surgical excision and primary fixation was performed at room temperature for 18 to 23 hours. The single large tumor was divided so that the single small tumor included up to the surrounding tissue, and the multiple masses were performed according to their individual sizes. Cases 1-5, 7, and 10-12 were small single tumors. Cases 6 and 13 were multiple masses. Cases 8, 9, 14, and 15 were single, large masses, 3 blocks from case 8, 4 blocks from case 9, 3 blocks from case 14, and 1 block from case 15, respectively.
（１）固定と切り出し
組織検体は外科切除後直ちに10％中性緩衝ホルマリンに浸漬し、室温下で18～23時間の一次固定を行った。単発性小型の腫瘤は周囲組織までを含むように、単発性大型の腫瘤は分割し、多発性腫瘤は、それぞれ個々の大きさに応じて行った。症例1～5、7、10～12は単発性小型腫瘤であった。症例6、13は多発性腫瘤であった。症例8、9、14、15は単発性大型腫瘤で、それぞれ症例8からは3ブロック、症例9からは4ブロック、症例14からは3ブロック、症例15からは1ブロック作成した。 1-2. Preparation procedure of immunostained specimen (1) Fixation and excision Tissue specimens were immersed in 10% neutral buffered formalin immediately after surgical excision and primary fixation was performed at room temperature for 18 to 23 hours. The single large tumor was divided so that the single small tumor included up to the surrounding tissue, and the multiple masses were performed according to their individual sizes. Cases 1-5, 7, and 10-12 were small single tumors. Cases 6 and 13 were multiple masses. Cases 8, 9, 14, and 15 were single, large masses, 3 blocks from case 8, 4 blocks from case 9, 3 blocks from case 14, and 1 block from case 15, respectively.
（２）包埋処理
切り出した固定標本は10%中性緩衝ホルマリンに再度浸漬し、室温下で30～31時間の二次固定を行い、自動固定包埋装置（ティシュー・テック VIRTM5ジュニア、code VIP-5-Jr-J0、サクラ精機株式会社）を用いて70%アルコール、80%アルコール、90%アルコール、95%アルコール、99%アルコールI、99%アルコールII、99%アルコールIIIを各2時間、キシレンI、キシレンII、キシレンIIIを各45分、パラフィンIを30分、パラフィンIIを30分、パラフィンIIIを45分、パラフィンIVを45分の順で浸透させた後、包埋皿に入れて、パラフィンブロックとして包埋した。包埋した組織は、3～5μmに薄切後、スライドグラス（MICRO SLIDE GLASS プレクリン水切放、code S-7224、松浪硝子工業）(MICRO SLIDE GLASS 水縁磨フロスト、code S-8215、松浪硝子工業)に張り付けて組織切片とし、免疫組織化学染色を実施した。 (2) Embedding treatment The excised fixed specimen is re-immersed in 10% neutral buffered formalin and subjected to secondary fixation at room temperature for 30 to 31 hours. Automatic fixation embedding device (Tissue Tech VIRTM5 Junior, code VIP -5-Jr-J0, Sakura Seiki Co., Ltd.) 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 99% alcohol I, 99% alcohol II, 99% alcohol III for 2 hours each Infiltrate xylene I, xylene II, xylene III for 45 minutes each, paraffin I for 30 minutes, paraffin II for 30 minutes, paraffin III for 45 minutes, and paraffin IV for 45 minutes in this order, and then put in an embedding dish. Embedded as a paraffin block. The embedded tissue is sliced to 3-5μm and then slide glass (MICRO SLIDE GLASS pre-clean water drainage, code S-7224, Matsunami Glass Industry) (MICRO SLIDE GLASS water edge polishing frost, code S-8215, Matsunami Glass Industry) ) To obtain tissue sections, and immunohistochemical staining was performed.
切り出した固定標本は10%中性緩衝ホルマリンに再度浸漬し、室温下で30～31時間の二次固定を行い、自動固定包埋装置（ティシュー・テック VIRTM5ジュニア、code VIP-5-Jr-J0、サクラ精機株式会社）を用いて70%アルコール、80%アルコール、90%アルコール、95%アルコール、99%アルコールI、99%アルコールII、99%アルコールIIIを各2時間、キシレンI、キシレンII、キシレンIIIを各45分、パラフィンIを30分、パラフィンIIを30分、パラフィンIIIを45分、パラフィンIVを45分の順で浸透させた後、包埋皿に入れて、パラフィンブロックとして包埋した。包埋した組織は、3～5μmに薄切後、スライドグラス（MICRO SLIDE GLASS プレクリン水切放、code S-7224、松浪硝子工業）(MICRO SLIDE GLASS 水縁磨フロスト、code S-8215、松浪硝子工業)に張り付けて組織切片とし、免疫組織化学染色を実施した。 (2) Embedding treatment The excised fixed specimen is re-immersed in 10% neutral buffered formalin and subjected to secondary fixation at room temperature for 30 to 31 hours. Automatic fixation embedding device (Tissue Tech VIRTM5 Junior, code VIP -5-Jr-J0, Sakura Seiki Co., Ltd.) 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 99% alcohol I, 99% alcohol II, 99% alcohol III for 2 hours each Infiltrate xylene I, xylene II, xylene III for 45 minutes each, paraffin I for 30 minutes, paraffin II for 30 minutes, paraffin III for 45 minutes, and paraffin IV for 45 minutes in this order, and then put in an embedding dish. Embedded as a paraffin block. The embedded tissue is sliced to 3-5μm and then slide glass (MICRO SLIDE GLASS pre-clean water drainage, code S-7224, Matsunami Glass Industry) (MICRO SLIDE GLASS water edge polishing frost, code S-8215, Matsunami Glass Industry) ) To obtain tissue sections, and immunohistochemical staining was performed.
（３）ヘマトキシリン・エオジン（HE）染色
組織切片は脱パラフィン処理（キシレンIを7分、キシレンIIを7分、99%アルコールIを5分、99%アルコールIIを10分、80%アルコールを7分、70%アルコールを7分）し、蒸留水に10回通した。次いでヘマトキシリン液（ティシュー・テックヘマトキシリン3G、code 8656、サクラファインテックジャパン）に3分、流水洗30分、蒸留水に10回通した。その後、エオジン液（ティシュー・テック エオジン、code 8659サクラファインテックジャパン）で22分間染色し、分色・脱水処理（70%アルコール、80%アルコール、90%アルコール、95%アルコールに各数回通し、99%アルコールI、99%アルコールII、99%アルコールIII、99%アルコールIVを各5分）と透徹処理（キシレンIを7分、キシレンIIを7分、キシレンIIIを10分）を行い、標本用封入剤（NEW M・X、code FX00500、松浪硝子工業を用いて封入した。 (3) Hematoxylin and eosin (HE) staining Tissue sections were deparaffinized (xylene I for 7 minutes, xylene II for 7 minutes, 99% alcohol I for 5 minutes, 99% alcohol II for 10 minutes, 80% alcohol for 7 minutes And 70% alcohol for 7 minutes) and passed through distilled water 10 times. Subsequently, the mixture was passed through a hematoxylin solution (Tissue Tech Hematoxylin 3G, code 8656, Sakura Finetech Japan) for 3 minutes, washed with running water for 30 minutes, and distilled water 10 times. Then, it is stained with eosin liquid (Tissue Tech eosin, code 8659 Sakura Finetech Japan) for 22 minutes, color separation and dehydration treatment (70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, each several times, 99% Alcohol I, 99% Alcohol II, 99% Alcohol III, 99% Alcohol IV for 5 minutes each) and thorough treatment (xylene I for 7 minutes, xylene II for 7 minutes, xylene III for 10 minutes) Encapsulant (NEW M • X, code FX00500, Matsunami Glass Industry Co., Ltd. was used.
組織切片は脱パラフィン処理（キシレンIを7分、キシレンIIを7分、99%アルコールIを5分、99%アルコールIIを10分、80%アルコールを7分、70%アルコールを7分）し、蒸留水に10回通した。次いでヘマトキシリン液（ティシュー・テックヘマトキシリン3G、code 8656、サクラファインテックジャパン）に3分、流水洗30分、蒸留水に10回通した。その後、エオジン液（ティシュー・テック エオジン、code 8659サクラファインテックジャパン）で22分間染色し、分色・脱水処理（70%アルコール、80%アルコール、90%アルコール、95%アルコールに各数回通し、99%アルコールI、99%アルコールII、99%アルコールIII、99%アルコールIVを各5分）と透徹処理（キシレンIを7分、キシレンIIを7分、キシレンIIIを10分）を行い、標本用封入剤（NEW M・X、code FX00500、松浪硝子工業を用いて封入した。 (3) Hematoxylin and eosin (HE) staining Tissue sections were deparaffinized (xylene I for 7 minutes, xylene II for 7 minutes, 99% alcohol I for 5 minutes, 99% alcohol II for 10 minutes, 80% alcohol for 7 minutes And 70% alcohol for 7 minutes) and passed through distilled water 10 times. Subsequently, the mixture was passed through a hematoxylin solution (Tissue Tech Hematoxylin 3G, code 8656, Sakura Finetech Japan) for 3 minutes, washed with running water for 30 minutes, and distilled water 10 times. Then, it is stained with eosin liquid (Tissue Tech eosin, code 8659 Sakura Finetech Japan) for 22 minutes, color separation and dehydration treatment (70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, each several times, 99% Alcohol I, 99% Alcohol II, 99% Alcohol III, 99% Alcohol IV for 5 minutes each) and thorough treatment (xylene I for 7 minutes, xylene II for 7 minutes, xylene III for 10 minutes) Encapsulant (NEW M • X, code FX00500, Matsunami Glass Industry Co., Ltd. was used.
（４）CD34の免疫組織化学染色
免疫組織化学染色はストレプトアビジンビオチン（SAB）法およびポリマー法を用いて行った。まず、組織切片は脱パラフィン処理（キシレンI、キシレンII、99%アルコールI、99%アルコールII、80%アルコール、70%アルコールを各10分）し、蒸留水に10回通した。次いでリン酸緩衝液（PBS）（リン酸緩衝液 20倍濃縮液 pH7.4 1/15mol/L、code 2S0481、関東化学）で5分、2回洗浄し、蒸留水に30秒通し、PBSで5分、1回洗浄した。内因性ペルオキシダーゼを阻止するために、余分な水分を取り除いた後、3%過酸化水素加メタノール（過酸化水素水、code18084-00、関東化学；99.8%メタノール、code25183-70、関東化学）に室温下で15分間浸した。その後PBSで5分、2回洗浄し、蒸留水に30秒通し、PBSで5分、1回洗浄した（以下、この過程をPBS洗浄と記載する）。抗原賦活化処理は圧力釜（ティファール クリプソクレール、型式P4310731、グループセブジャパン）を用い、切片は抗原賦活化液（Dako REALTM Target Retrieval Solution、code S2031、Dako）に浸して、5分間加圧処理した。加圧処理後に室温下で20分冷却し、その後、PBS洗浄を行った。 (4) Immunohistochemical staining of CD34 Immunohistochemical staining was performed using the streptavidin biotin (SAB) method and the polymer method. First, tissue sections were deparaffinized (xylene I, xylene II, 99% alcohol I, 99% alcohol II, 80% alcohol, and 70% alcohol for 10 minutes each) and passed 10 times through distilled water. Next, it was washed twice with phosphate buffer (PBS) (phosphate buffer 20-fold concentrated solution pH 7.4 1/15 mol / L, code 2S0481, Kanto Chemical) for 5 minutes, passed through distilled water for 30 seconds, and with PBS Washed once for 5 minutes. Remove excess water to block endogenous peroxidase, then add 3% hydrogen peroxide in methanol (hydrogen peroxide, code18084-00, Kanto Chemical; 99.8% methanol, code25183-70, Kanto Chemical) at room temperature Soaked for 15 minutes underneath. Thereafter, the plate was washed twice with PBS for 5 minutes, passed through distilled water for 30 seconds, and washed once with PBS for 5 minutes (this process is hereinafter referred to as PBS washing). Antigen activation treatment was performed using a pressure kettle (Tifar krypsocler, model P4310731, Group Cebu Japan), and the sections were immersed in the antigen activation solution (Dako REALTM Target Retrieval Solution, code S2031, Dako) and pressurized for 5 minutes. . After the pressure treatment, the mixture was cooled at room temperature for 20 minutes, and then washed with PBS.
免疫組織化学染色はストレプトアビジンビオチン（SAB）法およびポリマー法を用いて行った。まず、組織切片は脱パラフィン処理（キシレンI、キシレンII、99%アルコールI、99%アルコールII、80%アルコール、70%アルコールを各10分）し、蒸留水に10回通した。次いでリン酸緩衝液（PBS）（リン酸緩衝液 20倍濃縮液 pH7.4 1/15mol/L、code 2S0481、関東化学）で5分、2回洗浄し、蒸留水に30秒通し、PBSで5分、1回洗浄した。内因性ペルオキシダーゼを阻止するために、余分な水分を取り除いた後、3%過酸化水素加メタノール（過酸化水素水、code18084-00、関東化学；99.8%メタノール、code25183-70、関東化学）に室温下で15分間浸した。その後PBSで5分、2回洗浄し、蒸留水に30秒通し、PBSで5分、1回洗浄した（以下、この過程をPBS洗浄と記載する）。抗原賦活化処理は圧力釜（ティファール クリプソクレール、型式P4310731、グループセブジャパン）を用い、切片は抗原賦活化液（Dako REALTM Target Retrieval Solution、code S2031、Dako）に浸して、5分間加圧処理した。加圧処理後に室温下で20分冷却し、その後、PBS洗浄を行った。 (4) Immunohistochemical staining of CD34 Immunohistochemical staining was performed using the streptavidin biotin (SAB) method and the polymer method. First, tissue sections were deparaffinized (xylene I, xylene II, 99% alcohol I, 99% alcohol II, 80% alcohol, and 70% alcohol for 10 minutes each) and passed 10 times through distilled water. Next, it was washed twice with phosphate buffer (PBS) (phosphate buffer 20-fold concentrated solution pH 7.4 1/15 mol / L, code 2S0481, Kanto Chemical) for 5 minutes, passed through distilled water for 30 seconds, and with PBS Washed once for 5 minutes. Remove excess water to block endogenous peroxidase, then add 3% hydrogen peroxide in methanol (hydrogen peroxide, code18084-00, Kanto Chemical; 99.8% methanol, code25183-70, Kanto Chemical) at room temperature Soaked for 15 minutes underneath. Thereafter, the plate was washed twice with PBS for 5 minutes, passed through distilled water for 30 seconds, and washed once with PBS for 5 minutes (this process is hereinafter referred to as PBS washing). Antigen activation treatment was performed using a pressure kettle (Tifar krypsocler, model P4310731, Group Cebu Japan), and the sections were immersed in the antigen activation solution (Dako REALTM Target Retrieval Solution, code S2031, Dako) and pressurized for 5 minutes. . After the pressure treatment, the mixture was cooled at room temperature for 20 minutes, and then washed with PBS.
次いで余分な水分を取り除いた後、ブロッキング試薬として10%ウサギ正常血清（code 426052、ニチレイ）を切片に滴下し、室温下で10分、湿潤箱の中で反応させた。ブロッキング反応以降の抗体反応および発色の作業は全て湿潤箱内で行った。一次抗体は、免疫組織化学的にイヌとの交差性が知られている抗ヒトCD34ヤギポリクローナル抗体（sc-7045、SANTA CRUZ；Jennings et al., Vet Pathol., 49:532-537, 2012）を用いた。ブロッキング血清を流した後、一次抗体を滴下し、1:100の濃度で、4℃一晩（約12時間）反応させた。コントロールとして、ヤギIgG（PURIFIED GOAT IgG、code PCP001、AbD SEROTEC）を同じ濃度に希釈をして滴下した。反応後、KITPBS洗浄を行い、余分な水分を取り除いた。二次抗体としてヒストファインビオチン標識抗ヤギIgG抗体（code 416022、ニチレイ）を用いた。二次抗体は室温下で10分反応させた。酵素試薬としてヒストファインペルオキシダーゼ標識ストレプトアビジン（code 426062、ニチレイ）を滴下し、室温下で5分間反応させた。その後、PBS洗浄を行い、余分な水分を取り除いた。その後、シンプルステインDAB液（code 415172、ニチレイ）を滴下し、顕微鏡で確認のもと、7分間、発色を行った。蒸留水による発色停止後、マイヤーのヘマトキシリン（code 30002、武藤化学）にて30秒対比染色を行い、流水洗30分、蒸留水に10回通した。その後、脱水処理（70%アルコール、80%アルコール、90%アルコール、99%アルコールI、99%アルコールII、99%アルコールIIIを各7分）と透徹処理（キシレンI、キシレンII、キシレンIIIを各7分）を行い、M・Xを用いて封入した。
Then, after removing excess water, 10% rabbit normal serum (code 426052, Nichirei) as a blocking reagent was dropped onto the sections and reacted in a humid box at room temperature for 10 minutes. The antibody reaction and color development work after the blocking reaction were all carried out in a wet box. The primary antibody is an anti-human CD34 goat polyclonal antibody that is known to cross-react with dogs immunohistochemically (sc-7045, SANTA CRUZ; Jennings et al., Vet Pathol., 49: 532-537, 2012) Was used. After flowing blocking serum, the primary antibody was added dropwise and reacted at a concentration of 1: 100 overnight at 4 ° C. (about 12 hours). As a control, goat IgG (PURIFIED GOAT IgG, code PCP001, AbD SEROTEC) was diluted to the same concentration and added dropwise. After the reaction, KITPBS was washed to remove excess water. A histofine biotin-labeled anti-goat IgG antibody (code 416022, Nichirei) was used as a secondary antibody. The secondary antibody was reacted at room temperature for 10 minutes. As an enzyme reagent, histofine peroxidase-labeled streptavidin (code 426062, Nichirei) was added dropwise and reacted at room temperature for 5 minutes. Thereafter, PBS was washed to remove excess water. Thereafter, a simple stain DAB solution (code 415172, Nichirei) was added dropwise, and color development was performed for 7 minutes under a microscope. After stopping color development with distilled water, counterstaining was performed with Mayer's hematoxylin (code 30002, Muto Chemical) for 30 seconds, washed with running water for 30 minutes, and passed through distilled water 10 times. Then, dehydration treatment (70% alcohol, 80% alcohol, 90% alcohol, 99% alcohol I, 99% alcohol II, 99% alcohol III each for 7 minutes) and clearing treatment (xylene I, xylene II, xylene III each 7 minutes) and sealed with M · X.
１－３．病理組織学的評価
（１）HE染色
皮膚肥満細胞腫と診断された各症例の切片について、Patnaikの報告（Patnaik et al., Vet Pathol., 21:469-474, 1984）に従いグレード分類を行った。観察は、腫瘍の浸潤部位、細胞密度、細胞および核の形態、細胞境界、細胞質内顆粒の有無、高倍率視野中の核***像、好酸球浸潤の有無および程度、汗腺、リンパ管の拡張の有無および程度、膠原線維の変性の有無、壊死、浮腫および出血を含む間質反応について行った。腫瘍の浸潤部位を評価するうえで、表皮は角化層を持つ重層扁平上皮細胞で構成される層、真皮は毛包、脂腺、汗腺、リンパ管などが認められる、表皮下にある密線維性結合組織、皮下組織は脂肪組織を含む疎線維性結合組織とした。さらに、真皮を3層にわけ、表皮から付属器が存在する領域までの層を浅層、付属器が存在する領域を中層、中層より下層を深層とした。皮下組織は真皮に近い上層を浅層、筋層に近い下層を深層とした。 1-3. Histopathological evaluation (1) HE staining Grade sections of each case diagnosed as cutaneous mastocytoma were classified according to Patnaik's report (Patnaik et al., Vet Pathol., 21: 469-474, 1984). It was. Observations include tumor invasion site, cell density, morphology of cells and nuclei, cell borders, presence or absence of cytoplasmic granules, fission image in high magnification field, presence or absence of eosinophil infiltration, degree of sweat gland, lymphatic vessel dilation Interstitial reactions including presence and extent, presence or absence of collagen fiber degeneration, necrosis, edema and bleeding were performed. When evaluating the site of tumor invasion, the epidermis is a layer composed of stratified squamous epithelial cells with a keratinized layer, and the dermis is a dense fiber in the epidermis where hair follicles, sebaceous glands, sweat glands, lymphatic vessels, etc. are observed Sexual connective tissue and subcutaneous tissue were fibrillar connective tissue including adipose tissue. Furthermore, the dermis was divided into three layers, the layer from the epidermis to the region where the appendages were present was the shallow layer, the region where the appendages were present was the middle layer, and the layers below the middle layer were the deep layers. For the subcutaneous tissue, the upper layer close to the dermis was the shallow layer, and the lower layer close to the muscle layer was the deep layer.
（１）HE染色
皮膚肥満細胞腫と診断された各症例の切片について、Patnaikの報告（Patnaik et al., Vet Pathol., 21:469-474, 1984）に従いグレード分類を行った。観察は、腫瘍の浸潤部位、細胞密度、細胞および核の形態、細胞境界、細胞質内顆粒の有無、高倍率視野中の核***像、好酸球浸潤の有無および程度、汗腺、リンパ管の拡張の有無および程度、膠原線維の変性の有無、壊死、浮腫および出血を含む間質反応について行った。腫瘍の浸潤部位を評価するうえで、表皮は角化層を持つ重層扁平上皮細胞で構成される層、真皮は毛包、脂腺、汗腺、リンパ管などが認められる、表皮下にある密線維性結合組織、皮下組織は脂肪組織を含む疎線維性結合組織とした。さらに、真皮を3層にわけ、表皮から付属器が存在する領域までの層を浅層、付属器が存在する領域を中層、中層より下層を深層とした。皮下組織は真皮に近い上層を浅層、筋層に近い下層を深層とした。 1-3. Histopathological evaluation (1) HE staining Grade sections of each case diagnosed as cutaneous mastocytoma were classified according to Patnaik's report (Patnaik et al., Vet Pathol., 21: 469-474, 1984). It was. Observations include tumor invasion site, cell density, morphology of cells and nuclei, cell borders, presence or absence of cytoplasmic granules, fission image in high magnification field, presence or absence of eosinophil infiltration, degree of sweat gland, lymphatic vessel dilation Interstitial reactions including presence and extent, presence or absence of collagen fiber degeneration, necrosis, edema and bleeding were performed. When evaluating the site of tumor invasion, the epidermis is a layer composed of stratified squamous epithelial cells with a keratinized layer, and the dermis is a dense fiber in the epidermis where hair follicles, sebaceous glands, sweat glands, lymphatic vessels, etc. are observed Sexual connective tissue and subcutaneous tissue were fibrillar connective tissue including adipose tissue. Furthermore, the dermis was divided into three layers, the layer from the epidermis to the region where the appendages were present was the shallow layer, the region where the appendages were present was the middle layer, and the layers below the middle layer were the deep layers. For the subcutaneous tissue, the upper layer close to the dermis was the shallow layer, and the lower layer close to the muscle layer was the deep layer.
（２）CD34陽性細胞の評価
抗CD34抗体を用いた免疫組織化学的染色結果は、その染色強度によりJimenez et al., Vet Pathol., 49:532-537, 2000において報告されている評価方法に従い、評価した。具体的には、低倍率で染色切片を観察し、最も染色強度が強い領域を選出し、その領域を高倍率視野下で観察し、腫瘍性肥満細胞を100個計測した。100個それぞれの細胞の染色強度を分類し、それぞれの陽性率（%）を算出した。染色強度は以下の4つに分類した。
０：染色性が認められない、あるいはバックグラウンドと同程度の殆ど僅かに染色されているもの。
１＋：低倍率では0と区別がつかないが、高倍率で淡く染色性が確認されるもの。
２＋：低倍率で染色性が確認可能で、高倍率で完全に染色性が確認されるもの。
３＋：低倍率で完全に染色性が確認されるもの。 (2) Evaluation of CD34 positive cells The results of immunohistochemical staining using anti-CD34 antibody were determined according to the evaluation method reported in Jimenez et al., Vet Pathol., 49: 532-537, 2000, depending on the staining intensity. ,evaluated. Specifically, the stained sections were observed at a low magnification, a region having the strongest staining intensity was selected, the region was observed under a high magnification field, and 100 tumor mast cells were counted. The staining intensity of each of 100 cells was classified, and the positive rate (%) of each was calculated. The staining intensity was classified into the following four.
0: No staining property is observed, or almost the same level as the background.
1+: Indistinguishable from 0 at low magnification, but light and highly stainable at high magnification.
2+: The dyeability can be confirmed at a low magnification, and the dyeability can be completely confirmed at a high magnification.
3+: Staining property is completely confirmed at a low magnification.
抗CD34抗体を用いた免疫組織化学的染色結果は、その染色強度によりJimenez et al., Vet Pathol., 49:532-537, 2000において報告されている評価方法に従い、評価した。具体的には、低倍率で染色切片を観察し、最も染色強度が強い領域を選出し、その領域を高倍率視野下で観察し、腫瘍性肥満細胞を100個計測した。100個それぞれの細胞の染色強度を分類し、それぞれの陽性率（%）を算出した。染色強度は以下の4つに分類した。
０：染色性が認められない、あるいはバックグラウンドと同程度の殆ど僅かに染色されているもの。
１＋：低倍率では0と区別がつかないが、高倍率で淡く染色性が確認されるもの。
２＋：低倍率で染色性が確認可能で、高倍率で完全に染色性が確認されるもの。
３＋：低倍率で完全に染色性が確認されるもの。 (2) Evaluation of CD34 positive cells The results of immunohistochemical staining using anti-CD34 antibody were determined according to the evaluation method reported in Jimenez et al., Vet Pathol., 49: 532-537, 2000, depending on the staining intensity. ,evaluated. Specifically, the stained sections were observed at a low magnification, a region having the strongest staining intensity was selected, the region was observed under a high magnification field, and 100 tumor mast cells were counted. The staining intensity of each of 100 cells was classified, and the positive rate (%) of each was calculated. The staining intensity was classified into the following four.
0: No staining property is observed, or almost the same level as the background.
1+: Indistinguishable from 0 at low magnification, but light and highly stainable at high magnification.
2+: The dyeability can be confirmed at a low magnification, and the dyeability can be completely confirmed at a high magnification.
3+: Staining property is completely confirmed at a low magnification.
１－４．統計解析
上記の手順により得られた「CD34陽性細胞の評価」のデータについて、グレードとの関係について統計解析としてクラスカル・ワーリス検定を行い、検定の結果有意差が認められた場合、水準間の差を検定するために多重比較検定（Turkey-Kramer法）を行った。有意水準5%以下を有意差ありとした。
肥満細胞腫の転移の有無と生存日数との関係、及び、CD34の染色性と生存日数の関係について、ROC曲線（図１）を用いて陽性である染色性を判断し、ログランク検定を行った。ログランク検定は、JMPversion8.02を用いて行った。 1-4. Statistical analysis For the data of “Evaluation of CD34 positive cells” obtained by the above procedure, the Kruskal-Wallis test was performed as a statistical analysis on the relationship with grade, and if a significant difference was found as a result of the test, the difference between the levels A multiple comparison test (Turkey-Kramer method) was performed. A significance level of 5% or less was considered significant.
Using the ROC curve (Figure 1) to determine the positive staining for the relationship between the presence or absence of mastocytoma metastasis and the number of days of survival, and the relationship between CD34 staining and the number of days of survival, perform a log rank test. It was. The log rank test was performed using JMPversion 8.02.
上記の手順により得られた「CD34陽性細胞の評価」のデータについて、グレードとの関係について統計解析としてクラスカル・ワーリス検定を行い、検定の結果有意差が認められた場合、水準間の差を検定するために多重比較検定（Turkey-Kramer法）を行った。有意水準5%以下を有意差ありとした。
肥満細胞腫の転移の有無と生存日数との関係、及び、CD34の染色性と生存日数の関係について、ROC曲線（図１）を用いて陽性である染色性を判断し、ログランク検定を行った。ログランク検定は、JMPversion8.02を用いて行った。 1-4. Statistical analysis For the data of “Evaluation of CD34 positive cells” obtained by the above procedure, the Kruskal-Wallis test was performed as a statistical analysis on the relationship with grade, and if a significant difference was found as a result of the test, the difference between the levels A multiple comparison test (Turkey-Kramer method) was performed. A significance level of 5% or less was considered significant.
Using the ROC curve (Figure 1) to determine the positive staining for the relationship between the presence or absence of mastocytoma metastasis and the number of days of survival, and the relationship between CD34 staining and the number of days of survival, perform a log rank test. It was. The log rank test was performed using JMPversion 8.02.
２．結果
２－１．Patnaikの分類
15の症例に由来する検体について、HE染色標本を作製し、観察して、Patnaikの報告（上掲）に基づいて、イヌの皮膚肥満細胞腫のグレード分類を行った。
その結果を表２にまとめた。
２－２．抗CD34抗体の免疫組織学的染色の結果
肥満細胞腫の症例15例に由来する検体について、抗CD34抗体で染色し、その染色性に関し、＋２（「＋＋」）以上の染色性を示すものを「陽性」と判断した（図１、表２）。肥満細胞腫のPatnaikグレード3の染色性は、染色性＋１以上を陽性と判断した場合、グレード1および2と比較して、有意に陽性細胞率が高いことが明らかとなった（図２）。また、各検体の手術日から計測した生存日数、転移の有無について予後調査を行った。以上の結果を表２にまとめた。抗CD34抗体の染色性を示す典型的な染色像を図３に示す。 2. Result 2-1. Patnaik Classification For specimens from 15 cases, HE-stained specimens were prepared, observed, and graded for canine cutaneous mastocytoma based on Patnaik's report (supra).
The results are summarized in Table 2.
2-2. Results of immunohistochemical staining of anti-CD34 antibody Samples derived from 15 cases of mastocytoma were stained with anti-CD34 antibody, and the staining property was +2 (“++”) or higher. It was judged as “positive” (FIG. 1, Table 2). As for the staining property of Patnaik grade 3 of mastocytoma, it was revealed that the positive cell rate was significantly higher than that of grades 1 and 2 when staining property +1 or higher was judged positive (FIG. 2). In addition, the prognostic survey was conducted on the survival days measured from the operation date of each specimen and the presence or absence of metastasis. The above results are summarized in Table 2. A typical stained image showing the staining property of the anti-CD34 antibody is shown in FIG.
２－１．Patnaikの分類
15の症例に由来する検体について、HE染色標本を作製し、観察して、Patnaikの報告（上掲）に基づいて、イヌの皮膚肥満細胞腫のグレード分類を行った。
その結果を表２にまとめた。
２－２．抗CD34抗体の免疫組織学的染色の結果
肥満細胞腫の症例15例に由来する検体について、抗CD34抗体で染色し、その染色性に関し、＋２（「＋＋」）以上の染色性を示すものを「陽性」と判断した（図１、表２）。肥満細胞腫のPatnaikグレード3の染色性は、染色性＋１以上を陽性と判断した場合、グレード1および2と比較して、有意に陽性細胞率が高いことが明らかとなった（図２）。また、各検体の手術日から計測した生存日数、転移の有無について予後調査を行った。以上の結果を表２にまとめた。抗CD34抗体の染色性を示す典型的な染色像を図３に示す。 2. Result 2-1. Patnaik Classification For specimens from 15 cases, HE-stained specimens were prepared, observed, and graded for canine cutaneous mastocytoma based on Patnaik's report (supra).
The results are summarized in Table 2.
2-2. Results of immunohistochemical staining of anti-CD34 antibody Samples derived from 15 cases of mastocytoma were stained with anti-CD34 antibody, and the staining property was +2 (“++”) or higher. It was judged as “positive” (FIG. 1, Table 2). As for the staining property of Patnaik grade 3 of mastocytoma, it was revealed that the positive cell rate was significantly higher than that of grades 1 and 2 when staining property +1 or higher was judged positive (FIG. 2). In addition, the prognostic survey was conducted on the survival days measured from the operation date of each specimen and the presence or absence of metastasis. The above results are summarized in Table 2. A typical stained image showing the staining property of the anti-CD34 antibody is shown in FIG.
２－３．ログランク検定
まず、転移の有無と生存日数との相関について検討した。その結果、転移の有るものは、無いものに比べて、有意に生存日数が短かった。このことから、症例の検査結果が信頼できるデータであることが確認された（図４）
次に、CD34の染色性と生存日数の関係について検討した。染色陽性の症例は、陰性の症例より生存日数が短く、予後が悪いと判断することができる（図５）。今回検討した症例における転移の有無は、腫瘍切除後に明らかとなった症例も含まれており、初診時の段階で判別ができなかった症例もあった。
以上の結果から、CD34の染色性による分類が、Patnaikグレード分類よりも優れた予後の使用になるものと言える。 2-3. Log rank test First, the correlation between the presence of metastasis and the number of days of survival was examined. As a result, those with metastasis had significantly shorter survival days than those without metastasis. From this, it was confirmed that the test result of the case is reliable data (FIG. 4).
Next, the relationship between CD34 staining and survival days was examined. Staining-positive cases can be judged to have a shorter survival period and negative prognosis than negative cases (FIG. 5). The presence or absence of metastasis in the cases examined in this study included cases that became apparent after tumor resection, and there were cases where it was not possible to discriminate at the stage of initial examination.
From the above results, it can be said that the classification based on the staining property of CD34 is a better prognostic use than the Patnaik grade classification.
まず、転移の有無と生存日数との相関について検討した。その結果、転移の有るものは、無いものに比べて、有意に生存日数が短かった。このことから、症例の検査結果が信頼できるデータであることが確認された（図４）
次に、CD34の染色性と生存日数の関係について検討した。染色陽性の症例は、陰性の症例より生存日数が短く、予後が悪いと判断することができる（図５）。今回検討した症例における転移の有無は、腫瘍切除後に明らかとなった症例も含まれており、初診時の段階で判別ができなかった症例もあった。
以上の結果から、CD34の染色性による分類が、Patnaikグレード分類よりも優れた予後の使用になるものと言える。 2-3. Log rank test First, the correlation between the presence of metastasis and the number of days of survival was examined. As a result, those with metastasis had significantly shorter survival days than those without metastasis. From this, it was confirmed that the test result of the case is reliable data (FIG. 4).
Next, the relationship between CD34 staining and survival days was examined. Staining-positive cases can be judged to have a shorter survival period and negative prognosis than negative cases (FIG. 5). The presence or absence of metastasis in the cases examined in this study included cases that became apparent after tumor resection, and there were cases where it was not possible to discriminate at the stage of initial examination.
From the above results, it can be said that the classification based on the staining property of CD34 is a better prognostic use than the Patnaik grade classification.
本発明は、非ヒト動物の腫瘍の悪性度の評価方法、及び、予後を判断する方法及び該方法の使用に適するキットを提供するものである。非ヒト動物、特に、イヌ、ネコ等のペット動物等の腫瘍の予後を適切に判断することを可能にする本発明の方法は、動物医療の分野において、その実用化が大いに期待される。
The present invention provides a method for evaluating the grade of malignancy of a non-human animal tumor, a method for determining a prognosis, and a kit suitable for use of the method. The method of the present invention that makes it possible to appropriately determine the prognosis of tumors in non-human animals, particularly pet animals such as dogs and cats, is highly expected to be put to practical use in the field of animal medicine.
Claims (11)
- 非ヒト動物の腫瘍の悪性度を評価する方法であって、腫瘍細胞を含む対象動物由来の試料中の細胞に発現しているCD34を検出し、試料中に存在する細胞に対する該CD34を発現する細胞の割合を算出し、その割合に基づいて該腫瘍の悪性度を評価する方法。 A method for evaluating the malignancy of a tumor in a non-human animal, wherein CD34 expressed in cells in a sample derived from a target animal containing tumor cells is detected, and the CD34 is expressed relative to cells present in the sample A method of calculating the proportion of cells and evaluating the malignancy of the tumor based on the proportion.
- 前記CD34を発現している細胞の割合が、試料中に存在する細胞の２０％以上である場合に、該腫瘍の悪性度が高いと判断することを特徴とする請求項１に記載の方法。 The method according to claim 1, wherein when the proportion of cells expressing CD34 is 20% or more of the cells present in the sample, it is determined that the malignancy of the tumor is high.
- 前記CD34の発現を検出することが、免疫組織化学的染色法であることを特徴とする請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the detection of the expression of CD34 is an immunohistochemical staining method.
- 前記「CD34を発現する細胞」を、抗CD34抗体による細胞の染色性に基づいて評価することを特徴とする請求項３に記載の方法。 4. The method according to claim 3, wherein the “cell expressing CD34” is evaluated based on the staining property of the cell with an anti-CD34 antibody.
- 前記染色性が２＋である場合に、「CD34を発現する細胞」と評価することを特徴とする請求項４に記載の方法。 The method according to claim 4, wherein when the staining property is 2+, it is evaluated as "a cell expressing CD34".
- 前記非ヒト動物が、イヌ又はネコであり、前記腫瘍が皮膚肥満細胞腫であることを特徴とする請求項１乃至５のいずれかに記載の方法。 The method according to any one of claims 1 to 5, wherein the non-human animal is a dog or a cat, and the tumor is a cutaneous mastocytoma.
- 非ヒト動物の腫瘍の補助療法又は内科的療法の要否を決定するために使用することを特徴とする請求項１乃至６のいずれかに記載の方法。 The method according to any one of claims 1 to 6, wherein the method is used for determining whether or not an adjunct therapy or a medical therapy for a non-human animal tumor is necessary.
- 非ヒト動物の腫瘍の外科手術を行う場合の切除範囲を決定するために使用することを特徴とする請求項１乃至６のいずれかに記載の方法。 The method according to any one of claims 1 to 6, wherein the method is used for determining a resection range when performing a surgical operation on a tumor of a non-human animal.
- 非ヒト動物の腫瘍の予後の予測を行うために使用することを特徴とする請求項１乃至６のいずれかに記載の方法。 The method according to any one of claims 1 to 6, wherein the method is used for predicting a prognosis of a tumor of a non-human animal.
- 抗CD34抗体を含んでなる、非ヒト動物の腫瘍の悪性度を評価するための診断用キット。 A diagnostic kit for evaluating the malignancy of a non-human animal tumor, comprising an anti-CD34 antibody.
- 前記非ヒト動物がイヌ又はネコであり、前記腫瘍が皮膚肥満細胞腫であることを特徴とする請求項１０に記載の診断用キット。 The diagnostic kit according to claim 10, wherein the non-human animal is a dog or a cat, and the tumor is cutaneous mastocytoma.
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| WO2013056217A1 (en) * | 2011-10-14 | 2013-04-18 | The Ohio State University | Methods and materials related to ovarian cancer |
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