CN105372425A - Prostate cancer malignancy degree judging kit and method - Google Patents
Prostate cancer malignancy degree judging kit and method Download PDFInfo
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- CN105372425A CN105372425A CN201510474274.9A CN201510474274A CN105372425A CN 105372425 A CN105372425 A CN 105372425A CN 201510474274 A CN201510474274 A CN 201510474274A CN 105372425 A CN105372425 A CN 105372425A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- Urology & Nephrology (AREA)
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Abstract
The invention aims at providing a molecular mark technique serving as a novel diagnosis method except for Gleasons classification and TMN classification to provide a method which can still more precisely and easily judge the prostate cancer malignancy degree on the condition of using a material inspected and extracted through a cell needle instead of a materials picked out during an operation in the early stage before the operation by using a living tissue examination material and Gleasonas classification in a combination mode. The invention relates to a judging kit and method which are used for judging (diagnosing) the prostate cancer malignancy degree and predicting prognosis of a patient.
Description
The divisional application that the application is the applying date is on 02 06th, 2007, application number is 200780052524.3, denomination of invention is the application of " grade malignancy of prostate cancer judges kit and its method ".
Technical field
The present invention relates to the grade malignancy for judging (diagnosis) prostate cancer thus the judgement kit of the prognosis of prediction patient and criterion.
Background technology
LAT1 gene be cancer blastema because of, be separated (Kanai, Y. in 1998 by people such as Jin Jing, Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., Endou, H.:ExpressionCloningandCharacterizationofaTransporterfor LargeNeutralAminoAcidsActivatedbytheHeavyChainof4F2Antig en.J.Biol.Chem273 (37): 23629-23632,1998).LAT1 gene, coding has 12 transmembrane protein of the 44KD of L-type amino acid transporter function, when coexisting with 1 transmembrane protein 4F2hc (also referred to as CD98), display Na
+the activity of the L movement system of the classics of the large neutral amino acids such as the transhipment of dependent/non-dependent ground leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophane, methionine, histidine.LAT1 and 4F2hc albumen can be thought by cysteine-cysteine key and combine.LAT1 gene is proved and is deriving from the cultured cell of cancer, in tire liver, a large amount is expressed, in the normal tissue only at brain, placenta, testis, position expression (the Yanagida that marrow etc. are limited, O., Kanai, Y., Chairoungdua, A., Kim, D, Kyung., Segawa, H., Nii, T., Cha, S, Ho., Matsuo, H., Fukushima, J., Fukasawa, Y., Tani, Y., Taketani, Y., Uchino, H., Kim, J, Young., Inatomi, J., Okayasu, I., Miyamoto, K., Takeda, E., Goya, T., Endou, H.:HumanL-typeaminoacidtransporter1 (LAT1): characterizationoffunctionandezpressionintumorcelllines. BiochimicaetBiophysicaActa1514:291-302, 2001).On the other hand, 4F2hc gene then distributes widely in most normal structure and cancer cell etc.
Homolog LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., Kanai, the Y.:IdentificationandFunctionalCharacterizationofaNa of LAT1
+independentNeutralAminoAcidTransporterwithBroadSubstrate Selectivity.J.Biol.Chem274 (28): 19745-19751,1999) 50% amino acid identity is had with LAT1 albumen, about people's Tissue distribution of LAT2 gene, in the middle existence in a organized way of investigated institute.Therefore, LAT1 can be called the various L-type amino acid transporter of cancer embryo, and LAT2 can be called the various L-type amino acid transporter of normal type.4F2hc as the activate factor of LAT almost distributes in whole tissues, cancerous tissue and normal structure as broad as long.
But prostate cancer is the cancer that morbidity frequency is high viewed from world wide, and in the cancer of American male, morbidity rate is the 1st, and mortality ratio is the 2nd.Even if in Japan, patients with prostate cancer number also has the trend of increase in recent years, and the patient's number about 2,000 people between 1975 year then reported about 20,000 people in 2000, estimates will reach about 80,000 people at the year two thousand twenty, in the cancer of the male sex, be only second to lung cancer, morbidity rate reaches the 2nd.In addition, predict that the death toll caused by prostate cancer will reach present 1.4 times at the year two thousand twenty, estimate to become the 1st (Japan Clinic supplementary issue numbers 60 in U.S.'s mortality ratio; 44-48,2002; Prostate diseases clinical).
As the diagnosis of prostate cancer, the early detection of PSA value in blood, screening technique are famous, and are widely used, but the method does not have specificity to cancer, can raise when hypertrophy of the prostate, prostatitis even if known yet.In addition, in recent years, also report that PSA is only in proportion with prostatic simply in blood.Usually, if confirm, in blood, PSA value is high level, then confirm prostatic size, hardness etc. by rectal touch.When being suspected generation prostate cancer consumingly by above-mentioned inspection, extract living tissue and check, by haematoxylin Yihong (Hematoxylineosin) dyeing, carry out histopathologic diagnosis.In this diagnosis, by the state of the diagnosis cells such as degree of profile (gradeofatypism), degree of differentiation, judge its pernicious degree.In histopathologic diagnosis, Gleasons classification is the distinctive degree of profile classification of prostate cancer, be the method for carrying out classification according to the degree of degree of profile by 5 grades of scoring 1 ~ 5, in recent years, the method is just becoming determining that methods for the treatment of has the diagnosis of very large impact.
As the diagnosis using the material extracted during operation to carry out, the diagnosis based on TMN classification (T: primary tumo(u)r, N: lymphatic metastasis, M: far-end shifts) is considered to effective.
These diagnosises, particularly Gleasons classification, prognosis decision method as prostate cancer is widely used, itself and contacting of prognosis also have report, but present situation is in relevant field, in order to the Diagnosis and Treat that precision is more high, expect Gleasons classification, TMN by stages outside increase new diagnosis, particularly expect the appearance of molecular labeling.In addition, as mentioned above, the diagnosis of the material extracted when TMN classification etc. is as mentioned above and is used in operation, is therefore difficult to diagnose the grade malignancy of early-stage cancer.
[patent documentation 1] Japanese Unexamined Patent Publication 11-299489 publication
[patent documentation 2] Japanese Unexamined Patent Publication 2000-157286 publication
Therefore, the object of the invention is to, be provided in the method as the molecular labeling of new diagnosis outside Gleasons classification, TMN classification, there is provided by using the Gleasons sort merge of test alive material with commitment before surgery simultaneously, even if do not use the material extracted during operation and the material using cell needle check to extract time, also can easily judge the method for the grade malignancy of prostate cancer with more high precision.
Summary of the invention
For solving the problems of the technologies described above, first the present inventor etc. are conceived to amino acid transporter LATl specific expressed in the cultured cell deriving from cancer, tire liver.Then, using this LAT1 as molecular labeling, research make to find during the various gimmick of this manifesting of LAT1, even if someway use based on cell needle check extract sample time, for judging that the grade malignancy of prostate gland cancer cell is effective specifically, thus complete the present invention.
The present invention (1) be containing anti-human LAT1 monoclonal antibody, by immunohistochemical staining for judging the kit of the grade malignancy of prostate cancer.At this, as long as this anti-human LAT1 monoclonal antibody can identify that the antibody of LAT1 just has no particular limits specifically, such as, the antibody (such as mouse anti human LAT1 monoclonal antibody) of amino acid residue (MetAlaGlyAlaGlyProLysArgArgAlaLeuAlaAlaProAlaAlaGluGluLy sGluGluAlaArgGluLysMetLeuAlaAlaLysSerAlaAspGlySerAlaProA laGlyGluGlyGluGlyValThrLeuGlnArgAsnIleThrLeu) of 1 ~ 52 from the N distal portion of intracellular space identifying people LAT1 specifically can be enumerated.In addition, the amino acid sequence of people LAT1 and base sequence are recorded in Japanese Unexamined Patent Publication 2000-157286 publication.In addition, for " grade malignancy " in this instructions, the cancer of death the reason due to cancer is caused to be set as the cancer that grade malignancy is high, even if also can not be that the murderous cancer of immediate cause is set as the cancer that grade malignancy is low with cancer by being diagnosed as cancer.
At this, anti-human LAT1 monoclonal antibody, as long as be that antigen is combined with this antigen and just has no particular limits with LAT1, use mouse antibodies, rat Ab, rabbit antibody, goat-anti body etc. that can be suitable.
In addition, produce the hybridoma of monoclonal antibody, substantially can use known technology, make by the following stated.Namely can make by the following method: the cell of the antigen needed for required antigen, expression is used as sensitizing antigen, it is carried out immunity according to common immunization method, the immunocyte utilizing common Fusion of Cells method to make to obtain and known mother cell merge, utilize common screening method, screening produces the cell (hybridoma) of monoclonal antibody.The making of hybridoma, such as, can carry out according to the method for Milstein etc. (Kohler.G.andMilstein, C., MethodsEnzymol. (1981) 73:3-46) etc.When making anti-human LAT1 monoclonal antibody, the segment of LAT1 or its protein can be used as antigen, in addition, the cell of the segment expressing LAT1 or its protein can be used as antigen.In addition, the segment of LAT1 or its protein, such as, can obtain according to the method recorded in " " MolecuarCloning:ALaboratoryManual " the 2nd edition 1-3 rolls up the works such as Sambrook, J.; ColdSpringHarberLaboratoryPress publishes, 1989, New York ".In addition, the cell of expressing the segment of LAT1 or its protein also can obtain according to the method recorded in " " MolecuarCloning:ALaboratoryManual " the 2nd edition 1-3 rolls up Sambrook; the work such as J., and ColdSpringHarberLaboratoryPress publishes, 1989, New York ".
This kit, such as can containing other following inscapes.
(1) corresponding with the anti-human LAT1 monoclonal antibody antibody through peroxidase labelling
(2) superoxide (peroxide) of endogenic peroxidase is suppressed
(3) the redox pigment developed the color is caused by oxidation
(4) for making antigen protein (LAT1) and antibody be easy to the activating reagent combined
(5) retarding agent of the albumen beyond the LAT1 in suppression tissue and nonspecific combination of antibody
(6) in each step, removing is attached to the washing agent of the reagent of sample
Here, for the redox pigment of (3), there is multiple signal (such as fluorescence) that can carry out strength detection, but importantly can in visible region domain validation variable color.This is because, although principle is indefinite, when other signals, even if use the anti-human LAT1 monoclonal antibody that the present invention relates to, pernicious prostate cancer and optimum prostate cancer can not be distinguished clearly.On the other hand, the anti-human LAT1 monoclonal antibody that the application of the invention relates to and with at the agent combination of visible region domain validation variable color (immunohistochemical staining), just can distinguish pernicious prostate cancer and optimum prostate cancer clearly.
The present invention (2) be comprise anti-human LAT1 monoclonal antibody is applied to the operation of test sample tissue, based on the grade malignancy decision method of the prostate cancer of immunohistochemical staining.
At this, the method can comprise in other following operations any one or all.
Superoxide is applied to the operation of test sample tissue
Test sample is organized in activating reagent and floods, implement the operation of microwave treatment
Retarding agent is applied to the operation of test sample tissue
Apply the operation of the marking antibody corresponding with anti-human LAT1 monoclonal antibody
Apply the operation being caused the redox pigment developed the color by oxidation
According to circumstances, primary antibodie negative control is applied to the operation of test sample tissue
The present invention (3) comprise by the method for foregoing invention (2) judge the grade malignancy of prostate cancer operation and determine based on above-mentioned diagnostic result whether administration prostate treatment medicine operation, the method for differentiating to be suitable for the cases for prostate cancer that LAT1 molecular targeted therapy medicine uses.
Accompanying drawing explanation
Fig. 1 is the figure of the outward appearance of the dyeing of the prostate gland cancer cell that people's active somatic cell is shown.
Fig. 2 is the figure of the judgement example of the coloration result illustrated based on diagnosis of the present invention.
Fig. 3 is the figure of the result that specific test is shown.
Fig. 4 is the figure of the result that sensitivity test is shown.
Fig. 5 is the figure that the result that simultaneously repeatability (simultaneousrepeatability) is tested is shown.
Fig. 6 is the figure that the chromatic result confirmed based on the anti-LAT1 antibody employing various sample is shown.
Fig. 7 be illustrate employ Kaplan-Meire method LAT1 scoring in the figure of result of survival analysis.
Fig. 8 is the figure of LAT1 scoring and the life cycle relation of the method illustrated based on Si Ningkulaopu (ス ニ mono-Network ロ mono-プ).
Fig. 9 is the figure that the comparative result that PSA and LAT1 marks is shown.
Figure 10 is the figure that the comparative result of classifying with Stage is shown.
Figure 11 is the figure that the comparative result of marking with Gleasons is shown.
Embodiment
The diagnostic reagent of this embodiment is the in-vitro diagnosis kit of the propagation degree (grade malignancy) differentiating prostate gland cancer cell and diagnosis cancer cell.This kit contains the primary antibodie as the main reagent of reaction, comprises 6 kinds of reagent.Measuring principle is sketched, the LAT1 expressed in cancer cell, be there are 507 amino acid residues, have the function of the essential amino acid required for transmembrane transport cell proliferation, the functional protein of 12 cross-cell membrane types.The primary antibodie (anti-human/mouse monoclonal IgG antibody) used in the diagnosis of this embodiment is the antibody of amino acid residue of 1 ~ 52 from N distal portion of the intracellular space identifying LAT1 specifically.In addition, for this sequence, in the gene of people, had the albumen of identical sequence by ncbi database retrieval, result only retrieves LAT1.
The object of diagnostic reagent of the present invention checks sample, is that the formalin of prostate cancer biopsy material is fixed-histotomy of paraffin embedding sample.As detection method order, what use common confession pathological diagnosis fixes paraffin-embedded pathological tissue specimen through formalin, carries out with general immunohistochemical gimmick.That is, after paraffin-embedded sample and this antibody response, polymer reagent (two resist) and staining reagent will be used to dye, carry out observing (Fig. 1) with optical microscope.In addition, in Fig. 1, the position be colored is pernicious cancer cell.Be described the principle detected, anti-LAT1 antibody (primary antibodie) is incorporated on the LAT1 that expresses in cell membrane, and then forms complex with polymer reagent.And then the HRP of polymer reagent and chromogenic reagent react, in brown.This antibody is extremely strong with the compatibility of LAT1, when using this law, due to its high-sensitivity detection and selectivity, the discriminating from the cancer cell at initial stage (initial stage of LAT1 expresses) to the cancer of Late stage cancer cells (LAT1 overexpression) can be carried out, and semi-quantitatively can identify the expression of LAT1, as its application result, the progress extent (grade malignancy) of tumour can be evaluated.
[embodiment]
The Production Example > of < Production Example 1 primary antibodie
Mouse anti human L-type amino acid transporter 1 (hLAT1) monoclonal antibody is contained with 2 μ g/mL protein contents in primary antibodie.This antibody is, the albumen of the 1-52 position of the hLAT1 that In Vitro Translation (invitrotranslation) method will be adopted to be synthesized by hLAT1 cloning vector is as antigen, immunity is carried out to BALB/c mouse, its spleen cell and mouse bone marrow cells tumour cell are merged thus the hybridoma obtained, this hybridoma is inoculated in mouse peritoneal, from the ascites of gained, carry out purifying by ammonium sulfate fractionation separation and G-protein coupling column chromatography, then be dissolved in the antibody in the 10mMPBS (pH7.4) containing 1% bovine serum albumin(BSA).In addition, the amino acid sequence of LAT1 of encoding is recorded in Japanese Unexamined Patent Publication 2000-157286 publication with the base sequence of this protein.
< Production Example 2 judges the configuration example > of kit
The judgement kit of this Production Example, comprises 6 kinds of following reagent.
Retardance reagent
Normal swine serum is diluted to 2% thus modulation.
Primary antibodie
Little mouse-anti LAT1 monoclonal antibody (Production Example 1) is diluted to 2 μ g/mL thus modulation with damping fluid (1%BSA, 0.25% casein-sodium, 15mM sodium azide, 0.1% polysorbas20).
Polymer reagent
Use Nichirei HistofinesimplestainMAX-PO (M) (trade mark).In addition, this reagent contains the goat anti-mouse IgG polyclonal antibody (Fab ') of 4 μ g/mL peroxidase labellings.
Primary antibodie negative control
Mouse IgG (vector library (VectorLaboratories)) is dissolved in above-mentioned damping fluid, is deployed into 2 μ g/mL.
Matrix buffer
By trishydroxymethylaminomethane (Tris [hydroxylmethyl] aminomethane) and Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris [hydroxylmethyl] aminomethanechloride) pure water dilution, thus modulation.
Chromogenic substrate
By DAB (3-3 ' diaminobenzidine four hydrochloride (3-3 ' Diaminobendinetetrahydrochloride)) dissolve in damping fluid (above-mentioned Matrix buffer), furnishing 0.2mg/mL.
And then the judgement kit of this Production Example can containing the following reagent used in dyeing.
Endogenous peroxydase retardance reagent: 1%H
2o
2/ methyl alcohol
By aquae hydrogenii dioxidi methanol dilution to 1%.
Activating reagent: 0.01M citrate buffer solution (pH6.0)
Citric acid monohydrate compound (0.36g), trisodium citrate monocalcium salt compound (2.44g) are dissolved in distilled water, is modulated to 1L.
Cleansing solution: PBS
Sodium hydrogen phosphate 12 hydrate (2.90g), sodium dihydrogen phosphate dihydrate (0.296g), sodium chloride (8.5g) are dissolved in distilled water, is modulated into 1L.
If to arranging above, the formation reagent (required 6 kinds) of the diagnostic kit in this Production Example is the reagent shown in following table 1.
One example of the formation reagent of the diagnosis kit of this Production Example of table 1
< method of operating and decision method >
1. method of operating
The summary of operation technique shown in table 2.
Table 2 Immunohistochemical detection system
1-1. manual manipulation method
After test sample histotomy is taken off paraffin, impregnated in staining plate in endogenous peroxydase retardance reagent, after 30 minutes, wash in room temperature treatment.The remaining moisture of removing sample, is impregnated in activation reagent, then microwave treatment 5 minutes.After being fully cooled to room temperature after process, wash, wash with cleansing solution further.The remaining moisture of removing sample, the retardance reagent instilling q.s on tissue sections makes it intersperse among sample fully, room temperature reaction 30 minutes in moistening case.The remaining moisture of removing sample, the primary antibodie of instillation q.s, in moistening case, room temperature reaction is after 1 hour, carries out washing (5 minutes, 3 times) with cleansing solution.At negative control with in test sample histotomy, the primary antibodie negative control of instillation q.s, to replace primary antibodie, similarly processes.The remaining moisture of removing sample, the polymer reagent of instillation q.s, room temperature reaction 30 minutes in moistening case, carries out washing (5 minutes, 3 times) with cleansing solution.The remaining moisture of removing sample, instill a certain amount of matrix solution or impregnate matrix solution to test sample, in moistening case, or room temperature reaction, after 15 minutes, washs with cleansing solution in staining plate.By sample with after counterstain liquid (example: plum (Mayer) family name haematoxylin liquid) dyeing, wash.Dewater with alcohols, after dimethylbenzene displacement, sealing, for microscopy.
1-2. adopts the method for operating of active immunity dyeing apparatus
Test sample histotomy, retardance reagent, primary antibodie, primary antibodie negative control, polymer reagent, matrix solution, distilled water, cleansing solution, counterstain liquid are arranged at the position of the regulation of machine, make each reagent respectively with stipulated time, room temperature, react under moisture state.Remove the moisture of test sample with alcohols, then after replacing with dimethylbenzene, sealing, for microscopy.
2. criterion
After carrying out observing the overall colored state of confirmation with low range (4 times, object lens), in tumor tissues in test sample tissue, the part the highest to staining power is observed, by following scale (Fig. 2) with high magnification (10 times ~ 40 times, object lens).
A) 0 is marked
LAT1 overexpression: nothing
Dyeing pattern: (Fig. 2 a) for the cell in the LAT1 positive on the cell membrane of tumor tissues unconfirmed.
B) 1 is marked
LAT1 overexpression: nothing
Dyeing pattern: confirm that the cell in the LAT1 positive on the cell membrane in tumor tissues exists multiple, but staining power weak (Fig. 2 b).
C) 2 are marked
LAT1 overexpression: nothing
Dyeing pattern: the cell membrane in tumor tissues has multiple in the cell of the moderate LAT1 positive.Staining power is in the middle of scoring 1 with scoring 3 (Fig. 2 c).
D) 3 are marked
LAT1 overexpression: have
Dyeing pattern: the cell in the strong LAT1 positive on the cell membrane in tumor tissues exists multiple, can confirm coiled type dyeing (Fig. 2 d) clearly on the whole at the cell membrane of each tumour cell.
< test example 1 quality validation test >
To the contrast section for confirming the quality judging kit (from the cultured cell of the prostate cancer of display LAT1 protein overexpression, PC3 strain), a) specific test is carried out, b) sensitivity test and c) repeatability test simultaneously based on following method.In addition, in the later test example containing this test example, substantially implement with the condition recorded in above-mentioned < Production Example 2> and < criterion >, enforcement part is carried out for the employing condition different from these, illustrates by other modes.
A) specific test
Use the judgement kit of Production Example 2, contrast section (the paraffin section sample from the cultured cell PC3 of prostate cancer) is dyeed according to above-mentioned " method of operating ", compares/discusses the primary antibodie in judgement kit and the coloration result of primary antibodie negative control.Coloration result is implemented scoringization (table 3) according to the standard of above-mentioned " criterion " with 0 ~ scoring 3 of marking.In arbitrary test test sample kit, the result of contrast section is scoring 3 (Fig. 3).
Table 3 uses the coloration result of primary antibodie
According to above result, when using primary antibodie, observe the specific staining image corresponding with the LAT1 protein expression of each experimental control pathological tissue specimen, determine that the dye levels testing test sample is no problem.
B) sensitivity test
Use the judgement kit of Production Example 2, using the primary antibodie in judgement kit as comparing contrast, primary antibodie solution in test test sample kit is diluted the solution of 8 times by use with antibody diluent, standard section and contrast section are dyeed according to above-mentioned " method of operating ", primary antibodie in service test test sample kit and solution primary antibodie being diluted 8 times dye, and compare result/discuss.Coloration result is carried out scoring (table 4, table 5) according to the standard of above-mentioned " criterion " with 0 ~ scoring 3 of marking.When using the primary antibodie solution of normal concentration, the cell membrane of contrast section can confirm the staining power of scoring 3, but when use normal concentration primary antibodie antibody diluent is diluted the solution of 8 times, the staining power of cell membrane of contrast section goes down as 1 (Fig. 4) that mark.
Table 4 sensitivity test result
Use the situation of common primary antibodie
Table 5 uses the situation of primary antibodie 8 times of dilutions
Can confirm according to above result, when being used as kit to form the primary antibodie of the normal concentration of product, contrast section is all scoring 3, on the other hand, when the primary antibodie of normal concentration is diluted 8 times, LAT1 marks decline, therefore confirms this kit and has the range of sensitivity with presence or absence of the LAT1 protein expression that can judge in pathological tissue specimen.
C) repeatability test simultaneously
Use the judgement kit of Production Example 2, standard test sample histotomy and contrast section each difference 3 are side by side dyeed according to above-mentioned " method of operating ", coloration result is compared/discussed.Coloration result is carried out scoring (table 6) according to the standard of " criterion " with 0 ~ scoring 3 of marking.3 contrast sections all show same dye levels, are identical scoring (Fig. 5).
The result that table 6 uses same reagent box, dyeed by 3 section preparations made from same tissue block
(experiment 1)
According to the above results, when 3 same contrast sections are dyeed simultaneously, in 3 batches of arbitrary kit, all can confirm obvious specific dyeing, mark also identical, therefore distinguish repeatability simultaneously and difference between criticizing no problem.
< test example 2 uses the chromatic validation test > of cancerous tissue sample and cultured cell sample
Use the judgement kit of Production Example 2, the current test implementing that the dyeability in the biopsy material sample (be formalin is fixed, paraffin-embedded section preparation) of the cultured cell PC3 strain of the prostate cancer from target internal organs and prostate cancer is confirmed.In addition, as a reference, also validation test is carried out for the operation extraction material of the colorectal cancer of known LAT1 high expressed.In addition, this test example is implemented according to the condition recorded in above-mentioned < Production Example 2> and < criterion > substantially, and only the concentration of primary antibodie is tested by condition shown in following table 7.
Table 7 research object
Table 8 use the information of pathological tissue
* the immunohistochemistry staining method by employing an antigen liquid carries out the scoring judged
At table 9 and Fig. 6, result is shown.
Table 9 uses the chromatic confirmation based on anti-LAT1 antibody of various sample
According to above-mentioned result, in arbitrary sample, all can confirm the strong dyeing of coiled type on cancer cell membrane, the immunohistochemical staining by employing this antibody can being confirmed, the high expressed of LAT1 in cancer cell can be observed.
< test example 3 clinical testing >
Next, consider from proving of clinical medical viewpoint necessary, attempt developing this test.For the case receiving surgery for prostate cancer, be used in the biopsy sample that operation consent extracts, carried out the immunohistochemical staining of LAT1.
The judgement kit of Production Example 2 is used to dye by utilizing the section preparation of biopsy preparation of specimen, finally evaluate with the intensity of dark brown benzidine staining image (scoring 0 ~ 3), described biopsy sample, refer to save after bioptic sampling fix through formalin, paraffin-embedded tissue block.For these all cases, be divided into 4 groups by scoring 0 ~ 3, by represent with the longitudinal axis survival rate, with transverse axis represent through during (moon number) the Kaplan-Meier method of carrying out drawing carry out statistical study.Its result, for using the survival rate reaching full 5 years as the grade malignancy of the cancer of index, can confirm to there is significant difference between the case group and the case group of scoring 3 of scoring 0.In addition, although for the group significant difference unconfirmed of scoring 1,2, still show the survival rate depending on each scoring.Therefore, it is feasible for this method being used as the new technology in the grade malignancy diagnosis of cancer.
And then pointed out, when developing the specific antibody pharmaceuticals of LAT1 albumen in the future, and when finding specific depressant (low molecular compound) of LAT1/4F2hc function, these existence becoming the LAT1 of target (target molecule) are carried out to the function of quantification, for differentiating that the adaptation case of curative has serviceability.
1) enforcement body and case load
Enforcement body is enforcement body A and enforcement body B bis-mechanisms, and the detail of its case load is as shown in the table.Single cases is extracted out respectively, for test from each mechanism.
Table 10 tests enforcement body and case load
2) tissue used
-paraffin embedding sample fixed by the formalin of the prostate cancer biopsy material extracted by cell needle check
3) method
Among pathological tissue specimen, the case of stageII ~ stageIV is regarded as in the diagnosis of extracting out randomly through Histopathology, uses this kit to implement immunohistochemical staining according to above-mentioned " method of operating ".Described pathological tissue specimen refers to, the cases being diagnosed as prostate cancer in two test enforcement bodies nineteen eighty-two ~ 2000 between extracted by cell needle check, the formalin preserved fixes-paraffin embedding pathological tissue specimen.According to above-mentioned " criterion ", coloration result is observed, scoring, the correlativity for coloration result (LAT1 scoring) and 5 years survival rates uses Kaplan-Meier method to analyze.
(3) result
1) detail of case load and coloration result
For the case being diagnosed as prostate cancer, use the judgement kit of Production Example 2, when being investigated by the expression of immunohistochemical staining to the LAT1 albumen in tumour cell, according to case, the dyeing reaching scoring 0 ~ 3 can be confirmed.The detail of the LAT1 scoring in Liang Ge mechanism is with shown in following table.In addition, if the result of all cases is carried out difference classification by scoring, then enforcement body A and B all has the trend that the case of scoring 0 is maximum, 3 cases of marking are few.In addition, its dye levels is also almost identical, thus judge based in the dyeing of the judgement kit of Production Example 2 without inter-agency difference.
The scoring of LAT1 protein expression in table 11 prostate cancer tissue
| Experiment mechanism A | Experiment mechanism B | Add up to | |
| Scoring 0 | 87(53.0) | 5(71.0) | 92(48.2) |
| Scoring 1 | 34(20.7) | 0(0) | 34(19.9) |
| Scoring 2 | 25(15.2) | 2(28.6) | 27(15.8) |
| Scoring 3 | 18(11.0) | 0(0) | 18(10.5) |
| Amount to | 164 | 7 | 171 |
Unit: be the ratio (%) relative to number of samples in each mechanism in pathology number, ()
2) survival analysis in the LAT1 scoring of Kaplan-Meire method is employed
For 1) case, the result of Liang Ge mechanism is merged, the expression degree (LAT1 scoring) of the LAT1 albumen in tumour cell and the relation between the life cycle of case patient are utilized the method (nonparametric technique of Kaplan-Meier, Nonparametricmethod) carry out survival analysis, results verification has significant difference between LAT1 high expressed (LAT1 scoring 3) and low expression group (LAT1 scoring 0 ~ 2).Specifically, the result of P<0.001 is obtained between scoring 3 and scoring 0.As can be known from these results, in the group of LAT1 scoring 3, about half case is dead within 5 years, can confirm and mark 0 case group between have significant difference (Fig. 7).
3) transformational analysis of the LAT1 scoring of Si Ningkulaopu method is adopted
For 1) all cases, outside staining power, be divided into 3 grades to observe dyeing scope, classify by focal (less than 10%), partial (10 ~ 30%), diffuse (more than 30%).For each observations, based on the method for Si Ningkulaopu, the coefficient corresponding with dyeing scope (focal is 1, partial be 2, diffuse be 3) is multiplied by LAT1 scoring, conversion LAT1 scoring (Fig. 8).For these scorings of LAT1 through conversion, adopting with 2) identical Kaplan-Meier method carries out survival analysis, and results verification LAT1 marks and notable difference between life cycle.Can confirm in scoring more than 4, and have significant difference between scoring 0 from this result, particularly mark in the case of more than 6,90% is dead within 5 years.As previously discussed, pointing out LAT1 scoring (0 ~ 9) be converted to by using the intensity of marking to LAT1 to be multiplied by expression range factor (focal=1, partial=2, diffuse=3) to carry out survival analysis, the higher diagnosis of precision can be carried out.
(3) with the comparing of PSA
For identical case, compare with the PSA of the known specific mark as prostate cancer.In these 171 cases used, for 127 cases of PSA value recorded before perioperatively, using PSA value 4ng/mL and 10ng/mL as threshold value, use Kaplan-Meire method to carry out survival analysis, compare with the result of marking based on LAT1.In arbitrary threshold value, PSA value is used all to fail to confirm the difference between low expression group and high expressed group.During the LAT1 scoring using identical case to carry out is analyzed, confirm there is significant difference (Fig. 9) between scoring 0 group and scoring 3 groups.
(4) that classifies with Stage compares
In case group, according to information during operation, carry out stage classification at pathological TMN by stages with on the basis of the information relevant to the presence or absence of transfer during operation, carry out Kaplan-Meire survival analysis.Use Stage classification can not confirm the significant difference between 5 years survival rates, on the other hand, uses LAT1 classification, in arbitrary Stage, marks between 0 group and scoring 3 groups and all confirms there is significant difference (Figure 10).
(5) that marks with Gleasons compares
Utilize the pathological diagnosis report the test book of the biopsy material used in test, Gleasons scoring during investigation diagnosis, for 154 cases obtaining information, survival analysis is carried out by Kaplan-Meire method, when comparing with the result of marking based on LAT1, both almost obtain identical result (Figure 11).As previously discussed, present invention show and in prostate cancer diagnosis, be regarded as most important Gleasons mark the equal serviceability of the result (P=0.0008) of classifying.
(6) conclusion
According to this clinical test results, the expression of LAT1 and the life cycle of patients with prostate cancer inversely related significantly, point out its serviceability can mark with Gleasons and be equal to mutually.In addition, the present invention and the present mark as prostate cancer and the PSA that uses and for compared with representing that Stage that cancer progress degree uses classifies, also obtain good result in the grade malignancy diagnosis of cancer.
The possibility that industry utilizes
This method is good to the dyeing sensitivity of prostate tumor cells, although relevant to the characteristic of antibody, does not find non-specific responding, is also proved to be without inter-agency error.
In addition, by using this gimmick, the LAT1 that prostate tumor cells film is expressed amount is carried out visual qualitatively, prompting can the grade malignancy of diagnosis of prostate tumour cell, and then its serviceability is marked with Gleasons and is equal to, with PSA with Stage classify compare can think useful.And then this law is by immunohistochemical staining, and the LAT1 qualitatively on diagnosis cell film, classify with Gleasons, compared with other pathological diagnosis method, observation and result of determination easy.
And then wait in expectation and to combine by this gimmick and Gleasons mark, the grade malignancy of the prostate cancer making precision higher is diagnosed and is become possibility.Particularly in the diagnosis based on Gleasons classification, TMN classification, when Gleasons scoring amounts near 7, when accepting the judgement of T3N0 (so-called StageII) in addition, now there is the situation being difficult to the diagnosis carrying out prognosis, by with method of the present invention and use, helpful to these gray areas clear and definite.
Therefore, this method can be called and is, the grade malignancy of diagnosing prostate cancer, carries out prognosis judgement, the diagnosis medicine of the prostate cancer that the what is called of the treatment policy after simultaneously also determining is new.
Claims (3)
1. an anti-human LAT1 monoclonal antibody, it obtains for antigen with the albumen of 1 ~ 52 from N distal portion of hLAT1.
2. a grade malignancy judgement kit for prostate cancer, it contains anti-human LAT1 monoclonal antibody according to claim 1, and based on immunohistochemical staining.
3. the purposes of anti-human LAT1 monoclonal antibody according to claim 1 in the grade malignancy judgement kit manufacturing prostate cancer, the grade malignancy judgement kit of described prostate cancer is based on immunohistochemical staining.
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| CN200780052524A CN101680895A (en) | 2007-02-06 | 2007-02-06 | Kit for deciding degree of malignancy in prostate cancer and method of using the same |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050003490A1 (en) * | 1998-09-03 | 2005-01-06 | Japan Science And Technology Corporation | Neutral amino acid transporter and gene thereof |
| CN1630632A (en) * | 2002-02-07 | 2005-06-22 | 远藤仁 | Aromatic amino acid derivates and medicinal compositions |
| JP2005265531A (en) * | 2004-03-17 | 2005-09-29 | Tama Tlo Kk | Cancer detecting method using neutral amino acid transporter and kit therefor |
| WO2005121787A2 (en) * | 2004-06-04 | 2005-12-22 | Xenoport, Inc. | Lat1 transporters expressed in cancer cells |
| WO2005074996A3 (en) * | 2004-01-30 | 2006-01-05 | Xenoport Inc | Lat1 transporter expressed in blood brain barrier cells |
| JP2012163332A (en) * | 2011-01-28 | 2012-08-30 | J-Pharma Co Ltd | Kit for diagnosing malignancy of stomach cancer |
-
2007
- 2007-02-06 CN CN201510474274.9A patent/CN105372425A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050003490A1 (en) * | 1998-09-03 | 2005-01-06 | Japan Science And Technology Corporation | Neutral amino acid transporter and gene thereof |
| CN1630632A (en) * | 2002-02-07 | 2005-06-22 | 远藤仁 | Aromatic amino acid derivates and medicinal compositions |
| WO2005074996A3 (en) * | 2004-01-30 | 2006-01-05 | Xenoport Inc | Lat1 transporter expressed in blood brain barrier cells |
| JP2005265531A (en) * | 2004-03-17 | 2005-09-29 | Tama Tlo Kk | Cancer detecting method using neutral amino acid transporter and kit therefor |
| WO2005121787A2 (en) * | 2004-06-04 | 2005-12-22 | Xenoport, Inc. | Lat1 transporters expressed in cancer cells |
| JP2012163332A (en) * | 2011-01-28 | 2012-08-30 | J-Pharma Co Ltd | Kit for diagnosing malignancy of stomach cancer |
Non-Patent Citations (1)
| Title |
|---|
| BABU E ET AL.: "Identification of a Novel System L Amino Acid Transporter Structurally Distinct from Heterodimeric Amino Acid Transporters", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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