CN105891495A - Kit for predicting prognosis of esophagus cancer patient by combining 5-lipoxygenase pathway proteins - Google Patents
Kit for predicting prognosis of esophagus cancer patient by combining 5-lipoxygenase pathway proteins Download PDFInfo
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Abstract
The invention discloses a kit for detecting expression condition of a paraffin embedding operative specimen of an esophagus cancer patient and further predicting prognosis of the patient by combining 5-lipoxygenase metabolic pathway protein antibodies. The kit comprises three target protein antibodies: 5-LO, FLAP and LTA4H antibodies, and also comprises goat serum, 0.01M citrate repair liquid, 3% H2O2, a Polymer enhancer, a Polymer polymer, a DAB chromogenic reagent and PBS solution. Compared with single index detection, the kit for predicting the prognosis of the patient by combining 5-lipoxygenase metabolic pathway proteins has higher prediction effect, ranking only second to prediction ability of TNM staging. Moreover, the immunohistochemical ultrasensitive two-step method in the invention is a mature and reliable method which can be widely used in primary hospitals.
Description
Technical field
The present invention relates to associating 5-lipoxygenase pathway protein antibody and utilize the super quick type two step method of immunohistochemistry to go to detect patient with esophageal carcinoma mark
This expression and then the test kit of prediction patient's prognosis.
Background technology
Esophageal squamous cell carcinoma (Esophageal Squamous Cell Carcinoma, ESCC) is that a kind of common upper digestive tract of China is disliked
Property tumor, occupies the 4th in the mortality of malignant tumors of compatriots.The new of the whole world annual more than 40 ten thousand is sent out in esophageal carcinoma case, and more than half is sent out
Raw in China, China is also the esophageal carcinoma first state occurred frequently generally acknowledged in the world.The seventies in last century, implement malignant tumor in China general
Looking into, establishing the areas such as Cixian, hebei Province, Linxian, henan Province, Yangquan Shanxi, Sichuan Yanting, Chaozhou-Shantou region, Guangdong and Huaian, Jiangsu is that esophageal cancer in China six is the most occurred frequently
District.
The prognosis of Accurate Prediction patient with esophageal carcinoma treatment further for clinic and follow up a case by regular visits to significant.But, at present except international TNM
By stages outer also do not have a kind of simple and effective method that the prognosis of patient with esophageal carcinoma can be more accurately predicted.TNM predicts that the prognosis of patient is compared by stages
Loaded down with trivial details, need to evaluate exactly the intramural invasion of tumor, the number of metastatic lymph node, tumor with or without metastasis etc., and 5-of the present invention
The test kit of lipoxygenase metabolic pathway albumen associated prediction patient's prognosis has higher prediction effect than single Indexs measure, is only second to TNM and divides
The predictive ability of phase, and the super quick type two step method of immunohistochemistry that is based on of the present invention is a kind of mature and reliable, can be widely used at basic hospital
Method.In this test kit, the background introduction of 5-lipoxygenase metabolic pathway antibody protein is as follows.
The esophageal carcinoma is paid attention to inflammation relation day, and leukotriene is important proinflammatory factor, can activated leukocyte, promote mononuclear cell and blood
The adhesion of endothelial tube, increase vascular permeability.Our early-stage Study confirms, in esophageal squamous cell carcinoma 5-LO metabolic pathway, except 5-lipoxygenase
(5-LO) also has the table excessively of other two pherons 5-lipoxygenase activating protein (FLAP) and leukotriene A 4 hydrolase (LTA4H) outside process LAN
Reach, i.e. be there occurs exception by the whole metabolic pathway of arachidonic acid to leukotriene, promote generation and the development of esophageal squamous cell carcinoma.
5-LO, in addition to expressing in normal white cell, have also discovered the expression of 5-LO in the epithelial cell of many tumors, and at great majority just
Often in epithelial cell, 5-LO does not expresses.Although 5-LO report of high expressed in various malignant tumor is a lot, but about this high expressed and trouble
The relation of the life cycle of person is the most unclear, and the early-stage Study of the present invention finds 5-LO high expressed bad pre-with patient in human esophageal carcinoma
The most closely related.
FLAP albumen is a kind of embrane-associated protein, is positioned on nuclear membrane or endoplasmic reticulum.FLAP need to be combined with arachidonic acid and Semen arachidis hypogaeae four
Olefin(e) acid is supplied to 5-LO, thus activates 5-LO approach.Studies have reported that FLAP high expressed in breast carcinoma, be the independent prognostic factor of breast carcinoma.
The early-stage Study of the present invention finds FLAP high expressed closely related with the poor prognosis of patient in human esophageal carcinoma.
Arachidonic acid can be oxidized to 5-hydroxyl peroxide tetraenoic acid by 5-LO after activating completely, and generates leukotriene A (LTA4) through bioconversion,
The latter produces LTB4 under the effect of LTA4 hydrolytic enzyme (LTA4H).The research that LTA4H expresses in tumor tissues is the fewest.The early stage of the present invention is ground
Study carefully discovery LTA4H high expressed closely related with the poor prognosis of patient in human esophageal carcinoma.
Although above-mentioned every kind of albumen is respectively provided with the ability of prediction patient with esophageal carcinoma prognosis, but 3 kinds of albumen are joined together to predict patient with esophageal carcinoma
Prognosis then has higher judgement usefulness, is only second to internationally recognized TNM predictive ability by stages.
Summary of the invention
The technical problem to be solved is to provide a kind of 3 protein antibodies associated prediction esophageal carcinoma based on immunohistochemical method and suffers from
The test kit of person's prognosis.
Wherein, above-mentioned immunohistochemical method mainly detection neutral formalin is fixed and paraffin-embedded specimen.
Wherein, mentioned reagent box mainly includes 3 kinds of protein antibodies, is 5-LO, FLAP and LTA4H antibody respectively.
Wherein, mentioned reagent box also includes: lowlenthal serum, 0.01M citrate repair liquid, 3%H2O2, Polymer reinforcing agent, Polymer
Polymer, DAB colour reagent, PBS solution.
Accompanying drawing explanation
Fig. 1 is that 5-LO albumen expresses the Kaplan-Meier survival curve (X2=4.388, P=0.036) of crowd in difference.
Fig. 2 is that FLAP albumen expresses the Kaplan-Meier survival curve (X2=4.544, P=0.033) of crowd in difference.
Fig. 3 is that LTA4H albumen expresses the Kaplan-Meier survival curve (X2=5.006, P=0.025) of crowd in difference.
Fig. 4 is the Kaplan-Meier survival curve (X2=5.922, P=0.015) judging this crowd after 3 kinds of albumen are combined.
Fig. 5 is ROC curve (note: the area under curve formed after 3 kinds of albumen associatings is maximum, is 0.573).
Detailed description of the invention
We have collected the specimens from pri of the patient with esophageal carcinoma of 297 examples 2007-2011 and achieve wax stone.Below in conjunction with the accompanying drawings embodiment make into
One step describes in detail.
The super quick type two step method step of immunohistochemistry is as follows.
Specimen is after serial section, dimethylbenzene dewaxing, graded ethanol aquation, with the expression of super quick type two step method testing goal albumen, specifically side
Method is as follows.
(1) section is through 0.01M citrate repair liquid antigen retrieval, 3%H2O2After deactivating endogenous peroxydase, lowlenthal serum are closed,
Respectively with 4 DEG C of overnight incubation of 5-LO, FLAP, LTA4H antibody.
(2) develop the color through DAB after section is hatched respectively with Polymer reinforcing agent and Polymer polymer.
(3) section redyes core through haematoxylin, gradient alcohol dehydration, and dimethylbenzene is transparent and neutral gum mounting.
(4) section is all washed through PBS solution after completing above-mentioned often step.
All of immunostained section uses blind independently to mark through 2 Pathology Doctors 's.Tumor cell slurry occurs that brown yellow granule is considered as
Positive signal.Staining power is divided into 4 grades: 0, feminine gender；1, weak dyeing；2, moderate dyes；3, dye by force.Positive cell percent
It is divided into 5 grades: 0,0～5%；1,6～25%；2,26～50%；3,51～75%；4, >=76%.Each specimen
Gross score is drawn by tumor cell staining power and the two-part product of positive tumor cell percent, and scope is 0～12.Unite for convenience
Meter, we are divided into low expression and high expressed 2 groups all cases, and 1 represents low expression group, and 2 represent high expressed group, its cut-off value according to
X-tile software determines.5-LO:0～4 is divided into low expression group, and 5～12 are divided into high expressed group；FLAP:0～2 is divided into low expression
Group, 3～12 are divided into high expressed group；LTA4H:0～4 is divided into low expression group, and 5～12 are divided into high expressed group.
3 indexs join together to judge that the equation of patient with esophageal carcinoma prognosis is as follows.
Y=α × A+ β × B+ γ × C, wherein α, β, γ are coefficient, are returned by Cox and can draw numerical value, therefore equation becomes
For Y=0.345 × A+0.416 × B+0.287 × C.A, B, C are the expression status of 3 albumen in this example specimen, and 1 represents low expression,
2 represent high expressed.Arrange according to order from small to large after calculating the Y value of every example specimen, the Y value that the most centrally located case is corresponding
For cut-off value, i.e. 1.464.It is low danger group less than or equal to the case of this value, is high-risk group higher than the case of this value, after being so divided into 2 groups
Carry out single factor test statistical analysis again.In addition it is also possible to go, by drawing ROC curve, area under curve (AUC) that each albumen of comparison formed
Size.
The statistical procedures of data.
SPSS13.0 statistical software is used to process data.The expression of each albumen and the age of patient, sex, tumor size, knub position,
Tumor differentiation degree, intramural invasion, lymph node status, TNM by stages between relation use Chi-Square inspection；The table of each albumen
The relation reaching the life span with patient uses Kaplan-Meier survival analysis；The independent hazard factor of predicted impact patient's prognosis uses Cox ratio
Example risk regression model；The usefulness of every kind of albumen prediction patient with esophageal carcinoma prognosis uses ROC curve；P ＜ 0.05 is considered there is significance,statistical.
Result shows: all by stages and lymph node status is closely related with the TNM of patient with esophageal carcinoma in the expression of 3 albumen.
Cox risk regression model detect the Risk value after each albumen is expressed alone or in combination and be whether the independent hazard factor of patient's prognosis such as
Under.
The expression status of 5-LO is closely related with the life span of patient, and also is independent prognostic factor (Risk=1.412,95%CI
=1.014-1.967, P=0.041).
The expression status of FLAP is closely related with the life span of patient, but is not independent prognostic factor (Risk=1.515,95%CI
=0.99-2.322, P=0.055).
The expression status of LTA4H is closely related with the life span of patient, and also be independent prognostic factor (Risk=1.333,95%
CI=1.023-1.737, P=0.033).
3 albumen are combined and are had higher esophageal carcinoma prognosis prediction ability, be also simultaneously independent prognostic factor (Risk=1.464,95%
CI=1.051-2.038, P=0.024), but inferior to TNM (Risk=1.878,95%CI=1.346-2.62, P ＜ 0.001) by stages
Predictive ability with lymph node status (Risk=1.922,95%CI=1.373-2.69, P ＜ 0.001).
The ROC curve area formed after 3 albumen associatings is only second to TNM by stages, higher than the predictive value of single each gene expression.
To sum up, it is the independent risk factor (P=0.024) evaluating patient with esophageal carcinoma prognosis after 5-LO, FLAP, LTA4H associating, less than it
Corresponding P value (respectively 0.041,0.055,0.033) when individually evaluating patient.
The Kaplan-Meier survival curve drawn after 3 index associatings individually evaluates the Kaplan-Meier curve drawn than each index
Difference becomes apparent from, and X2 value is maximum (X2=5.922), and P value is minimum (P=0.015).
Under the ROC curve obtained after 3 index associatings, area is 0.573, and the ROC being all higher than being formed when every kind of index is individually evaluated is bent
Area under line, is only second to the area under curve (0.618) that TNM is formed by stages.Therefore 5-LO, FLAP, LTA4H associating post-evaluation esophagus is said
The prognosis of cancer patient has a higher predictive value, and the most more preferably operates than TNM and the suitability is higher.
When evaluating the prognosis information of individual patient with esophageal carcinoma clinically, only need to be the SABC mark of these obtained 4 protein expressions of patient
Corresponding expression status numerical value (1 represents low expression, and 2 represent high expressed) is inserted in formula Y=0.345 × A+0.416 × B+0.287 × C,
Just can draw Y value, then compare with the cut-off value 1.464 that we determined that, belong to low danger group less than the patient of this value, existence in its 1 year
Rate is 86.8%, and within 2 years, survival rate is 65.9%, and within 3 years, survival rate is 28%；And belonging to high-risk group more than the patient of this value, its 1 year survival rate is
77.4%, within 2 years, survival rate is 57.4%, and within 3 years, survival rate is 19.1%.
Claims (10)
1. the test kit predicting patient with esophageal carcinoma prognosis, it is characterised in that test kit includes anti-5-lipoxygenase (5-LO), anti-5-fat oxygenate
Ras GTPase activating protein ras-GTP (FLAP), three kinds of protein antibodies of anti-leukotriene A 4 hydrolase (LTA4H).
The test kit of prediction patient with esophageal carcinoma prognosis the most according to claim 1, it is characterised in that: test kit also includes lowlenthal serum,
0.01M citrate repair liquid, 3%H2O2, Polymer reinforcing agent, Polymer polymer, DAB colour reagent, PBS solution.
The test kit the most according to claim 1 and 2 application in the test kit preparing associated prediction patient with esophageal carcinoma prognosis.
Application the most according to claim 3, it is characterised in that the specimen using test kit to be detected is that neutral formalin is fixed and paraffin embedding
Paraffin embedded tissues.
5. according to the application described in claim 3 or 4, it is characterised in that: the detection method used is the super quick type two step method of immunohistochemistry.
6. according to the application described in any one of claim 3-5, it is characterised in that the method for prediction patient with esophageal carcinoma prognosis comprises the steps: specimen
Sliced, dewaxing, after aquation with the expression of super quick type two step method testing goal albumen.
Application the most according to claim 6, it is characterised in that prediction patient with esophageal carcinoma prognosis method specific as follows: cut into slices through dewaxing to water,
After antigen retrieval, deactivating endogenous peroxydase and lowlenthal serum are closed, hatched in moisture preservation box with anti-5-LO, FLAP, a LTA4H respectively
Night；Nucleus is redyed through DAB colour developing and haematoxylin after hatching with Polymer reinforcing agent and Polymer polymer the most respectively；Through wine after
Loss of essence water and the transparent rear neutral gum mounting of using of dimethylbenzene, and every immunohistochemical staining section is marked.
8. according to the application described in any one of claim 3-7, it is characterised in that: the cut-off value of every kind of protein expression height is soft by X-tile
Part determines, wherein the cut-off value of 5-LO is 4 points, and the cut-off value of FLAP is 2 points, and the cut-off value of LTA4H is 4 points.
9. according to the application described in any one of claim 3-8, it is characterised in that: often example specimen is after 3 kinds of antibody of application detect respectively, its 3 kinds
The computing formula of albumen Combined expression is Y=α × A+ β × B+ γ × C, and wherein α, β, γ are coefficient, can be returned by Cox and draw,
Therefore this formula is converted to Y=0.345 × A+0.416 × B+0.287 × C；A, B, C are respectively 5-LO, FLAP, LTA4H egg in this example specimen
White expression status, 1 represents low expression, and 2 represent high expressed；Arrange according to order from small to large after the Y value calculating every example specimen, its
In the centrally located Y value corresponding to specimen be cut-off value, i.e. 1.464, then be low danger group less than or equal to the case of this value,
It it is high-risk group higher than the case of this value.
10. according to the application described in any one of claim 3-9, it is characterised in that: when evaluating the information of individual patient with esophageal carcinoma prognosis, only need to be this
The expression status corresponding to SABC score of 3 albumen of patient is inserted in formula, it is possible to draw Y value, then with we determined that
Cut-off value 1.464 compares, and belongs to low danger group less than or equal to the patient of this value, and its 1 year survival rate is 86.8%, and within 2 years, survival rate is 65.9%,
Within 3 years, survival rate is 28%, and belongs to high-risk group more than the patient of this value, and its 1 year survival rate is 77.4%, and within 2 years, survival rate is 57.4%, 3 years
Survival rate is 19.1%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107236791A (en) * | 2017-05-09 | 2017-10-10 | 赤峰学院 | The SNP and detection kit of one earlier evaluations esophageal squamous cell carcinoma onset risk |
| CN108732350A (en) * | 2017-04-20 | 2018-11-02 | 中国科学院上海生命科学研究院 | PLA2G6 is as tubercle and the biomarker of early warning liver cancer in instruction liver |
| CN108732351A (en) * | 2017-04-20 | 2018-11-02 | 中国科学院上海生命科学研究院 | EPS8L2 is as tubercle and the biomarker of early warning liver cancer in instruction liver |
| CN108732349A (en) * | 2017-04-20 | 2018-11-02 | 中国科学院上海生命科学研究院 | LTA4H is as tubercle and the biomarker of early warning liver cancer in instruction liver |
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| US20120283117A1 (en) * | 2005-05-03 | 2012-11-08 | Children's Hospital Medical Center | Determination of eosinophilic esophagitis |
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Patent Citations (2)
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| US20120283117A1 (en) * | 2005-05-03 | 2012-11-08 | Children's Hospital Medical Center | Determination of eosinophilic esophagitis |
| CN103765220A (en) * | 2011-08-29 | 2014-04-30 | 东丽株式会社 | Marker for detecting colorectal cancer or esophageal cancer and method for inspecting same |
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| HONG QIU等: "express of 5-lipoxygenase and leukotriene A4 hydrolase in hunman atherosclerotic lesions correlates with symptoms of plaque instability", 《PNAS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732350A (en) * | 2017-04-20 | 2018-11-02 | 中国科学院上海生命科学研究院 | PLA2G6 is as tubercle and the biomarker of early warning liver cancer in instruction liver |
| CN108732351A (en) * | 2017-04-20 | 2018-11-02 | 中国科学院上海生命科学研究院 | EPS8L2 is as tubercle and the biomarker of early warning liver cancer in instruction liver |
| CN108732349A (en) * | 2017-04-20 | 2018-11-02 | 中国科学院上海生命科学研究院 | LTA4H is as tubercle and the biomarker of early warning liver cancer in instruction liver |
| CN107236791A (en) * | 2017-05-09 | 2017-10-10 | 赤峰学院 | The SNP and detection kit of one earlier evaluations esophageal squamous cell carcinoma onset risk |
| CN107236791B (en) * | 2017-05-09 | 2020-09-25 | 赤峰学院 | SNP (single nucleotide polymorphism) for early evaluation of esophageal cancer onset risk and detection kit |
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