CN105717082A - Method for detecting heterogeneity in triple-negative breast cancer - Google Patents
Method for detecting heterogeneity in triple-negative breast cancer Download PDFInfo
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Abstract
The invention discloses a method for detecting heterogeneity in the triple-negative breast cancer.According to the method, tissue samples on different portions of the same patient are cured on the same tissue chip, and then the tissue chip is subjected to multiple kinds of detection such as immunohitochemistry, immunofluorescence and fluorescence in situ hybridization.Experimental cost is effectively reduced, time and labor are saved, errors caused by graded experiments can be reduced, the consistency of experimental results is improved, and good quality control is achieved.According to the method, heterogeneity in the triple-negative breast cancer is evaluated through the multi-probe combined fluorescence in situ hybridization technology, dependence on expensive equipment and invested cost of the expensive equipment are reduced, and large-scale popularization is easy.Heterogeneity in the triple-negative breast cancer is quantitatively detected, and definite evaluation indexes are provided for accurately predicting postoperative recurrence and transfer risks of the patient.
Description
Technical field
The present invention relates to Prognosis in Breast Cancer predictor field, particularly relate to method heterogeneous in detection by quantitative patient with breast cancer's tumor, specifically, be a kind of for detecting method heterogeneous in three negative breast carcinoma.
Background technology
Breast carcinoma is one of common cancer.According to WHO recent statistics data, China newly sends out Female breast cancer patients about 180,000 every year, occupies female malignant sickness rate second, and its treatment and prognosis prediction are subject to everybody extensive concern.
Breast carcinoma is the disease that a kind of heterogeneity is significantly high, and the factor such as its prognosis and different subtype (ER, PR, HER-2 receptor status), neoplasm staging, proliferation index (Ki-67), histological grade is closely related.But when factors above is consistent, it being, particularly with ER, PR, HER-2, the breast carcinoma hypotype that negative three negative breast cancer (TNBC) this prognosis is worst, the prognosis between different patients still suffers from difference.Therefore people are constantly exploring new, effective prognosis prediction index, accurately to judge Patients on Recurrence and to shift risk, adjust therapeutic strategy in time.
Except breast cancer treatment and the prognosis of different subtype there are differences, inside tumor also has significantly high heterogeneity, and there are some researches show, heterogeneous closely related with the diagnosis of disease, treatment and prognosis in the tumor of tumor.Such as, the tumor tissues fluorescence in situ hybridization detection HER-2 amplification of same patient's different parts is likely to inconsistent, the formulation of this result being likely to affect Clinical detection and therapeutic scheme.Therefore the heterogeneity assessing breast carcinoma is particularly important.But, method heterogeneous in detection by quantitative tumor tumor still ununified at present, and current most of research method has the disadvantage that
1. for this special heterogeneity of inside tumor, clinic often needs multi-point sampling, and then single sample carries out the analysis of gene molecule level respectively, and whole process time cost, Financial cost, human cost all increase significantly, also can increase experimental error simultaneously.
2. method heterogeneous in tumor of probing into relatively advanced at present includes full genome express spectra, full-length genome or exon order-checking, what even have carries out unicellular order-checking, above method Financial cost and technology require high, popularize on a large scale and are difficulty with, and lack practicality.
3. judge, by heterogeneous in tumor, the standard that tumor prognosis is ununified at present, and be not yet applied in three negative breast cancer.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, it is an object of the invention to provide that a kind of cost is low, error is little, many group samples can be detected simultaneously for detecting device and method heterogeneous in three negative breast carcinoma.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
A kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: comprise the following steps:
The making of heterogeneous detection chip in step one, three negative breast carcinoma:
1) acceptor wax block is made:
Under paraffin wax embedding, prepare acceptor wax block with mould；Adopt tissue sampling needle to punch on acceptor wax block, between adjacent holes core, there is certain interval, make Kong Xin form microarray on acceptor wax block；
2) donor wax stone is drawn materials:
Collect three negative breast cancerous tissues of the required detection of array and match normal structure paraffin specimen as donor wax stone；Often all carry out H&E dyeing after the section of group donor wax stone system, the typical breast carcinoma district of tissues observed structure, and iris out on slide, compare H&E stained, donor wax stone corresponding site makes labelling；Make on instrument at organization chip and with the sampling probe of same diameter, donor wax stone labelling position is sampled, the cancerous tissue of one group of peek diverse location and the tissue core of a normal control tissue；
3) in tumor, heterogeneous organization chip makes:
By often organizing the tissue core obtained on donor wax stone by rule of necessarily arranging, vertically fill in the blank well core of acceptor wax block correspondence position；Being put back to by ready-made organization chip in wax stone mould, organization chip, to fritting, is cooled down, is again heated to fritting, repeats for several times by heating, makes tissue core and acceptor wax block combine closely；Then by the equating of organization chip surface；Organization chip is cut into slices and carries out H&E dyeing, reaffirm draw materials a little whether identical with the target area of donor wax stone labelling；
Step 2, multiprobe associating fluorescence in situ hybridization:
1) probe selects: adopt two double-color probe and a monochromatic probe to carry out detection heterogeneous in three negative breast carcinoma；Each probe individually carries out fluorescence in situ hybridization, and step is identical, specific as follows:
A, section pretreatment: the organization chip section that will make, the white tiles insulation that will cut, overnight；Next day dewaxes in room temperature dimethylbenzene, then section is embathed process, naturally dries slide；
B, degeneration hybridization: drip probe under lucifuge and cover section target area, add a cover coverslip and use resin glue edge sealing, putting into hybridization instrument overnight；
C, washing are redyed: taking out slide under lucifuge, throw off sealing, room temperature is dipped in SSC liquid and makes coverslip naturally come off, and slide is embathed process, naturally dries slide, drip DAPI mounting, in fluorescence microscopy Microscopic observation and take pictures after placement 15min；
Heterogeneity in step 3, calculating tumor
1) each counting is multiple clear-cut does not have overlapping, fluorescence signal cell clearly with peripheral cell, records the number of each cell interior danger signal and green, and each cell can calculate its copy ratio；
2) information often organizing cell being collected, calculate heterogeneity index in the tumor often organized, concrete formula is:
H’=-∑piln(pi),
Wherein pi represents the ratio that the cell of i-th copy value is shared in all cells；Often the H ' of three probes is sued for peace and draws heterogeneity index H in total tumor by group.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
The acceptor wax block made in step one is of a size of the organization chip of 25.0mm × 23.8mm × 5.0mm for preparation, tissue sampling needle diameter is 1.0mm, the tissue sampling needle degree of depth of punching on acceptor wax block is about 0.4cm, the spacing of each two adjacent holes core is about 2.0mm, prepares into the microarray of 8 row × 8 row.
Step one is collected three negative breast cancerous tissues of 16 groups of required detections and matches normal structure paraffin specimen as donor wax stone, when donor wax stone labelling position is sampled, the tissue core of one group of cancerous tissue taking three diverse locations and a normal control tissue, four samples are from left to right arranged adjacent in same a line successively on acceptor wax block, and 16 groups of tissue cores fill up the microarray that 8 row × 8 of acceptor wax block arrange.
Step one is organized core plug enter after in the blank well core of acceptor wax block, ready-made organization chip is put back in wax stone lead stamp, put into 56 DEG C of oven heat 20min to fritting, organization chip is being taken out at room temperature natural cooling, place into oven heat, repeat the above steps 3 times, finally with paraffin slicing machine by the equating of organization chip surface.
Two double-color probe adopted in step 2 are EGFR/CEP7 probe and CCND1/CEP11 probe, and monochromatic probe is MYC probe.
The step of pretreatment of cutting into slices in step 2 is: the organization chip section that will make, and the white tiles cut is placed in 56 DEG C of baking boxs overnight；Dewax in room temperature dimethylbenzene next day 10min × 2 time, successively dehydrated alcohol 5min, and each 3min of graded ethanol 100%, 85%, 70%, distilled water 5min embathes；Pressure method 2min, gastric enzyme working solution 37 DEG C digestion 9-15min；Room temperature 2 × SSC solution 5min, 1% paraformaldehyde/1 × PBS10min, 2 × SSC solution 5min, each 3min of graded ethanol 75%, 85%, 100% embathes, and naturally dries slide.
In step 2, the probe amount of degeneration hybridization dropping is 10 μ L, degeneration 83 DEG C in hybridization instrument, and 8min hybridizes 42 DEG C, 16h.
Washing counterstaining step in step 2 is: taking out slide under lucifuge, throw off sealing, room temperature is dipped in 2 × SSC liquid and makes coverslip naturally come off, 37 DEG C of 50% Methanamide/2 × SSC15min successively, 2 × SSC15min, 0.1%NP-40/2 × SSC5min, each 3min of graded ethanol 75%, 85%, 100% embathes.Naturally dry slide, drip DAPI mounting, in fluorescence microscopy Microscopic observation and take pictures after placing 15min.
In step 3, each counting 100 is clear-cut does not have overlapping, fluorescence signal cell clearly with peripheral cell.
This invention address that the effect of technical problem:
1. being solidificated in by the tissue samples of same group of different parts on same organization chip, a chip can carry 15-20 group specimen simultaneously, and then this organization chip can carry out the multiple detections such as SABC, immunofluorescence, fluorescence in situ hybridization.Thus, we are capable of organizing tissue specimen more, the tissue specimen of same group of different parts carries out high-throughout detection simultaneously, not only reduce and can be effectively reduced experimental cost, saving time, manpower, also can reduce owing to the error caused is tested in gradation, improve the concordance of experimental result, it is achieved preferable quality controls.
2. adopting multiprobe associating fluorescence in situ hybridization technique to assess in three negative breast carcinoma first heterogeneous, relative in conventional tumor, heterogeneous detection method, reduces the dependency to expensive device and cost of investment, it is easy to promote on a large scale.
3. utilize multiprobe associating fluorescence in situ hybridization that heterogeneity in three negative breast carcinoma is carried out detection by quantitative first, it is proposed that the numerical range determined, for three negative breast carcinoma recurrences and the evaluation index providing clear and definite that shifts risk.
Accompanying drawing explanation
Fig. 1 is the structural representation of organization chip of the present invention.In figure, multiple round dots at middle part are tissue core bore.
Detailed description of the invention
The present invention's is a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: comprise the following steps:
One, the making of heterogeneous detection chip in three negative breast carcinoma:
1. make acceptor wax block:
(1) under paraffin wax embedding, the acceptor wax block used by 25.0mm × 23.8mm × 5.0mm chip is made with plumbous molding jig.
(2) adopting diameter is that 1.0mm tissue sampling needle punches on acceptor wax block, and the degree of depth is about 0.4cm, and the spacing of each two adjacent tissue core is about 2.0mm, prepares into the microarray of 8 row × 8 row.
2. donor wax stone is drawn materials:
(1) collect the cancerous tissue of 16 three Breast Cancer Patients with Negative Axillaries of required detection and match normal structure paraffin specimen as donor wax stone.Donor wax stone derives from conventional archive wax stone.
(2) carry out H&E dyeing after the section of donor wax stone system, pathologists examine under a microscope the typical breast carcinoma district of organizational structure, and iris out on slide.Comparison H&E stained, performs labelling on donor wax stone corresponding site.
(3) with the sampling probe of same diameter, donor wax stone labelling position is sampled on organization chip making instrument.
3. in tumor, heterogeneous organization chip makes:
(1) the tissue core obtained on donor wax stone is vertically filled in the blank well core of acceptor wax block correspondence position.Every patient takes the cancerous tissue (b, c, d) of a normal control tissue (a) and three diverse locations.A, b, c, d are from left to right arranged adjacent in same a line successively on acceptor wax block.
(2) ready-made organization chip is put back in wax stone lead stamp, put into 56 DEG C of oven heat 20min to fritting, period close observation chip status.Organization chip is taken out at room temperature natural cooling.Place into oven heat, repeat the above steps 3 times, it is therefore an objective to make tissue core and acceptor wax block combine closely.Finally with paraffin slicing machine by the equating of organization chip surface.
(3) organization chip being fabricated to cut into slices and carry out H&E dyeing, pathologists examining under a microscope, reaffirm draw materials a little whether identical with the target area of donor wax stone labelling, whether pathological diagnosis result consistent.
Two, multiprobe associating fluorescence in situ hybridization
(1) probe selects: according to previous experiments basis, and we combine two double-color probe of EGFR/CEP7, CCND1/CEP11 and mono-monochromatic probe of MYC carries out detection heterogeneous in three negative breast carcinoma.Each probe individually carries out fluorescence in situ hybridization, and step is identical, (2) specific as follows-(4).
(2) section pretreatment: the organization chip section that will make, is placed in 56 DEG C of baking boxs overnight by the white tiles cut.Dewax in room temperature dimethylbenzene next day 10min × 2 time, successively dehydrated alcohol 5min, and each 3min of graded ethanol 100%, 85%, 70%, distilled water 5min embathes.Pressure method 2min, gastric enzyme working solution 37 DEG C digestion 9-15min.Room temperature 2 × SSC solution 5min, 1% paraformaldehyde/1 × PBS10min, 2 × SSC solution 5min, each 3min of graded ethanol 75%, 85%, 100% embathes, and naturally dries slide.
(3) degeneration hybridization (lucifuge operation): drip 10 μ L probe coverage goal regions, add a cover coverslip and use resin glue edge sealing, put into hybridization instrument overnight (degeneration 83 DEG C, 8min hybridize 42 DEG C, 16h).
(4) (lucifuge operation) is redyed in washing: taking out slide, carefully throw off sealing, room temperature is dipped in 2 × SSC liquid and makes coverslip naturally come off, 37 DEG C of 50% Methanamide/2 × SSC15min successively, 2 × SSC15min, 0.1%NP-40/2 × SSC5min, each 3min of graded ethanol 75%, 85%, 100% embathes.Naturally dry slide, drip DAPI mounting, in fluorescence microscopy Microscopic observation and take pictures after placing 15min.
Three, calculate in tumor heterogeneous
(1) each counting 100 is clear-cut does not have overlapping, fluorescence signal cell clearly with peripheral cell, recording the number (MYC only need to record danger signal for monochromatic probe) of each cell interior danger signal and green, each cell can calculate its copy ratio (danger signal number/green number) (MYC is the number that monochromatic probe only need to count danger signal point).
(2) information of three point (b, c, d) totally 300 cells that every patient derives from tumor tissues collects, and calculates heterogeneity index (Shannonindex) in the tumor of every patient, and concrete formula is:
H’=-∑piln(pi),
Wherein pi represents the cell of i-th copy (ratio) value ratio shared by all cells.The H ' of three probes is sued for peace and draws heterogeneity index H in total tumor by every patient.According to result of calculation, if H<5.0, think that heterogeneity index H is low in tumor, if H>=5.0, think that heterogeneity index H is high in tumor.After the height of H index and this patient's breast carcinoma improvement excision, in 3 years, the risk of recurrence and transfer is directly proportional.
Below being only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment, and all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that, for those skilled in the art, some improvements and modifications without departing from the principles of the present invention, should be regarded as protection scope of the present invention.
Claims (9)
1., for detecting a method heterogeneous in three negative breast carcinoma, it is characterized in that: comprise the following steps:
The making of heterogeneous detection chip in step one, three negative breast carcinoma:
1) acceptor wax block is made:
Under paraffin wax embedding, prepare acceptor wax block with mould；Adopt tissue sampling needle to punch on acceptor wax block, between adjacent holes core, there is certain interval, make Kong Xin form microarray on acceptor wax block；
2) donor wax stone is drawn materials:
Collect three negative breast cancerous tissues of the required detection of array and match normal structure paraffin specimen as donor wax stone；Often all carry out H&E dyeing after the section of group donor wax stone system, the typical breast carcinoma district of tissues observed structure, and iris out on slide, compare H&E stained, donor wax stone corresponding site makes labelling；Make on instrument at organization chip and with the sampling probe of same diameter, donor wax stone labelling position is sampled, the cancerous tissue of one group of peek diverse location and the tissue core of a normal control tissue；
In tumor, heterogeneous organization chip makes:
By often organizing the tissue core obtained on donor wax stone by rule of necessarily arranging, vertically fill in the blank well core of acceptor wax block correspondence position；Being put back to by ready-made organization chip in wax stone mould, organization chip, to fritting, is cooled down, is again heated to fritting, repeats for several times by heating, makes tissue core and acceptor wax block combine closely；Then by the equating of organization chip surface；Organization chip is cut into slices and carries out H&E dyeing, reaffirm draw materials a little whether identical with the target area of donor wax stone labelling；
Step 2, multiprobe associating fluorescence in situ hybridization:
Probe selects: adopt two double-color probe and a monochromatic probe to carry out detection heterogeneous in three negative breast carcinoma；Each probe individually carries out fluorescence in situ hybridization, and step is identical, specific as follows:
Section pretreatment: the organization chip section that will make, the white tiles insulation that will cut, overnight；Next day dewaxes in room temperature dimethylbenzene, then section is embathed process, naturally dries slide；
B, degeneration hybridization: drip probe under lucifuge and cover section target area, add a cover coverslip and use resin glue edge sealing, putting into hybridization instrument overnight；
C, washing are redyed: taking out slide under lucifuge, throw off sealing, room temperature is dipped in SSC liquid and makes coverslip naturally come off, and slide is embathed process, naturally dries slide, drip DAPI mounting, in fluorescence microscopy Microscopic observation and take pictures after placement 15min；
Heterogeneity in step 3, calculating tumor
1) each counting is multiple clear-cut does not have overlapping, fluorescence signal cell clearly with peripheral cell, records the number of each cell interior danger signal and green, and each cell can calculate its copy ratio；
2) information often organizing cell being collected, calculate heterogeneity index in the tumor often organized, concrete formula is:
H’=-∑piln(pi),
Wherein pi represents the ratio that the cell of i-th copy value is shared in all cells；Often the H ' of three probes is sued for peace and draws heterogeneity index H in total tumor by group.
2. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: the acceptor wax block made in step one is of a size of the organization chip of 25.0mm × 23.8mm × 5.0mm for preparation, tissue sampling needle diameter is 1.0mm, the tissue sampling needle degree of depth of punching on acceptor wax block is about 0.4cm, the spacing of each two adjacent holes core is about 2.0mm, prepares into the microarray of 8 row × 8 row.
3. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: step one is collected three negative breast cancerous tissues of 16 groups of required detections and matches normal structure paraffin specimen as donor wax stone, when donor wax stone labelling position is sampled, the tissue core of one group of cancerous tissue taking three diverse locations and a normal control tissue, four samples are from left to right arranged adjacent in same a line successively on acceptor wax block, and 16 groups of tissue cores fill up the microarray that 8 row × 8 of acceptor wax block arrange.
4. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: step one is organized core plug enter after in the blank well core of acceptor wax block, ready-made organization chip is put back in wax stone lead stamp, put into 56 DEG C of oven heat 20min to fritting, organization chip is being taken out at room temperature natural cooling, placing into oven heat, repeat the above steps 3 times, finally with paraffin slicing machine by the equating of organization chip surface.
5. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: two double-color probe adopted in step 2 are EGFR/CEP7 probe and CCND1/CEP11 probe, monochromatic probe is MYC probe.
6. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: the step of pretreatment of cutting into slices in step 2 is: the organization chip section that will make, the white tiles cut is placed in 56 DEG C of baking boxs overnight；Dewax in room temperature dimethylbenzene next day 10min × 2 time, successively dehydrated alcohol 5min, and each 3min of graded ethanol 100%, 85%, 70%, distilled water 5min embathes；Pressure method 2min, gastric enzyme working solution 37 DEG C digestion 9-15min；Room temperature 2 × SSC solution 5min, 1% paraformaldehyde/1 × PBS10min, 2 × SSC solution 5min, each 3min of graded ethanol 75%, 85%, 100% embathes, and naturally dries slide.
7. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: in step 2, the probe amount of degeneration hybridization dropping is 10 μ L, degeneration 83 DEG C in hybridization instrument, 8min hybridizes 42 DEG C, 16h.
8. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: washing counterstaining step in step 2 is: take out slide under lucifuge, throw off sealing, room temperature is dipped in 2 × SSC liquid and makes coverslip naturally come off, 37 DEG C of 50% Methanamide/2 × SSC15min successively, 2 × SSC15min, 0.1%NP-40/2 × SSC5min, each 3min of graded ethanol 75%, 85%, 100% embathes；Naturally dry slide, drip DAPI mounting, in fluorescence microscopy Microscopic observation and take pictures after placing 15min.
9. according to claim 1 a kind of for detecting method heterogeneous in three negative breast carcinoma, it is characterized in that: in step 3, each counting 100 is clear-cut does not have overlapping, fluorescence signal cell clearly with peripheral cell.
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