CN107058565A - The diagnosis and treatment of acra type melanoma - Google Patents
The diagnosis and treatment of acra type melanoma Download PDFInfo
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Abstract
The invention belongs to medical field, it is related to the diagnosis and/or treatment preparation and its application of a kind of acra type melanoma.The invention provides the gene marker of auxiliary diagnosis and/or treatment acra type melanoma, the mark includes gene C DK4, CCND1 and P16INK4a.Copy number change of the present invention based on acra type melanoma and CDK4 passageway related genes has certain relation, using CDK4 pathway inhibitors as the treatment new strategy of acra type melanoma, provides for the personalized medicine of acra type melanoma and instructs foundation.
Description
Technical field
The invention belongs to medical field, it is related to the diagnosis and/or treatment preparation and its application of a kind of acra type melanoma.
Background technology
Melanoma is the malignant tumour caused by melanocyte canceration, in recent years, and obvious increase is presented in its incidence of disease
Trend.According to site of pathological change and by sun damage degree, malignant mela noma can be divided into four kinds of hypotypes：Chronic sun damage type
(CSD), non-chronic sun damage type (non-CSD), acra type (AM) and mucosal pattern.Due to the difference of genetic background, white people
Melanoma is using non-acra cutaneous melanoma as principal mode, and the disease rates of acra type and mucosal pattern melanoma only account for 5% He
1%；And yellow race's Humanmachine tumour, using acra type and mucosal pattern as principal mode, both ratios account for the 70% of melanoma.
Acra type melanoma generally occurs in palm, sole and nail.Compared with non-acra cutaneous melanoma, acra
Type melanoma has stronger invasion, and often prognosis is worse by patient.At present, the conventional chemotherapy of acra type melanoma, exempt from
Epidemic disease treatment, targeted therapy still lack the international treatment specification of system, and this may be less with acra type Proportion of patients in clinical research
It is relevant.In melanoma therapy field, selectively targeted treatment now for BRAF, C-KIT gene mutation and with PD-1,
CTLA-4 largely improves the therapeutic effect of advanced melanoma for the immunotherapy of representative.But it is black in acra type
In melanoma, BRAF, C-KIT gene mutation frequency are only 15.5% and 11.9%.It is, thus, sought for it is new, effectively control
Target spot is treated to treat acra type melanoma.
The content of the invention
There is provided a kind of new based on the important driving gene copy number variation analysis to acra type melanoma patients by the present invention
Therapy target and preparation, diagnosis for acra type melanoma and treatment provide a kind of new approach.
In the first aspect, the present invention provides a kind of auxiliary diagnosis and/or treats the genetic marker of acra type melanoma
Thing, the mark includes gene C DK4 (NG_007484.2RefSeqGene)；Further, in addition to CCND1 (NG_
007375.1RefSeqGene) or P16INK4a(NG_007485.1RefSeqGene)；Or the mark includes gene
CCND1 and P16INK4a。
Preferably, described gene C DK4, CCND1 and P16INK4aIn at least one there is copy number change.The copy
Number change is the number change compared with normal type or wild type.
In second aspect, a kind of acra type melanoma auxiliary diagnosis of present invention offer and/or medicine for treatment are instructed
Multiple gene detection kit, the kit is used for the gene marker for detecting the present invention.Preferably, the kit is used for
Detect the copy number of gene marker.
Preferably, the kit includes detection gene C DK4, CCND1 and P16INK4aProbe；It is further preferred that institute
Stating the article No. that probe is Applied Biosystems companies is respectively：CDK4:HS02225231_CN、P16INK4a:
HS03717141_CN and CCND1:HS03803699_CN probe.The PCR detection system is used as reference using Rnase P genes
Gene, no nucleotides water replaces template to be negative control, and normal skin DNA is used as normal reference.PCR reaction systems：20 μ L, bag
Include：The μ L of Genotyping Master Mix 10, Copy Number Target Assay 1
μL、Copy Number Reference Assay (Rnase P) 1 μ L, DNA profiling 80ng/ holes, double distilled water
Supply (8-DNA templates volume).
Preferably, the kit includes detection gene C DK4, CCND1 and P16INK4aPcr amplification primer thing.
At the 3rd aspect, the present invention provides the gene marker in the preparation auxiliary related to acra type melanoma
Application in the preparation or kit of diagnosis and/or treatment.
At the 4th aspect, the present invention provides a kind of preparation for being used to treat acra type melanoma, and the preparation is included
CDK4 pathway inhibitors.
At the 5th aspect, the present invention provides CDK4 pathway inhibitors in the medicine for preparing treatment acra type melanoma
Application.
In the present invention, it is preferred to, the acra type melanoma is the acra type melanoma that CDK4 copy numbers are expanded；
It is further preferred that the acra type melanoma is CDK4 copy number amplifications+P16INK4aThe acra type black of copy number reduction
Plain knurl, the acra type melanoma of CDK4 copy number amplification+CCND1 copy numbers amplification；Or for CCND1 copy numbers amplification+
P16INK4aThe acra type melanoma of copy number reduction.
Preferably, the CDK4 pathway inhibitors are AT7519, and its structure is shown in formula I：
AT7519 is ATP competitiveness CDK inhibitor, when acting on CDK1,2,4,6 and 9, IC50For 10-210nM；To CDK3
Action effect is slightly weak, to CDK7 almost without inhibitory activity.When acting on cell, animal, cell propagation, tumour life can be suppressed
It is long.Carry out I/II phase clinical researches in the tumours such as Huppert's disease, leukaemia, melanoma clinical research is not yet opened
Exhibition.
The present invention is by studying CDK4 paths, it was found that acra type melanoma and CDK4 passageway related genes
Copy number change has certain relation, and this prompting targeting cell-cycle path is probably one of acra type melanoma and important controlled
Target spot is treated, important research evidence is provided applied to acra type melanoma for CDK4 inhibitor, as acra type melanoma
Treat new strategy.Meanwhile, the present invention demonstrates different CDK4 pathway inhibitors to be had for the acra type melanoma of different partings
There are different curative effects.When patient be diagnosed as suffer from acra type melanoma when, further detection gene C DK4, CCND1 and
P16INK4aCopy number, so that it is determined that using which kind of therapeutic agent.This provides finger for the personalized medicine of acra type melanoma
Lead foundation.
Brief description of the drawings
Fig. 1：Influence of the CDK4 pathway inhibitors of various dose to the cell propagation of different genotype；Wherein, A is suppression
Agent AT7519, B are that inhibitor LY2835219, C are LEE011；
Fig. 2：In the mouse model of different genotype, the curve map of influences of the AT7519 to mouse tumor volume；Wherein A~
F is respectively experimental group and control group curve map in different PDTX models, and A is PDX-001, and B is PDX-006, and C is PDX-012, D
For PDX-015, E is PDX-017, and F is PDX-016；
Fig. 3：The pictorial diagram of influences of the AT7519 to mouse tumor volume in the mouse model of different genotype.
Embodiment
Present invention, but the scope of the present invention not limited to this will be further illustrated in following examples.Unless specific instructions,
The reagent and instrument used in following examples is all this area conventional reagent and instrument, can be obtained by commercially available approach；Institute
The experimental method used is normal experiment method, and those skilled in the art can know how to repeat institute completely according to prior art
State and test and obtain accordingly result.
In the present invention, CCND1 is writing a Chinese character in simplified form for gene C yclin D1, and the two is used interchangeably；P16 is Gene PTENINK4a
Write a Chinese character in simplified form, the two is used interchangeably.
Embodiment one：Genetic mutation is verified
428 acra type melanoma patients tumor tissues paraffin sections are collected altogether (derives from Beijing Tumour Hospital's kidney
428 acra type melanoma patients of melanoma internal medicine registration, patient's signature informed consent form, voluntarily provide its paraffin bag
The melanoma tumor histotomy buried is used for this research), QuantiGene Plex (QGP) methods and real-time glimmering are used respectively
Fluorescent Quantitative PCR (Sonde method) detection CDK4 passageway related genes-CDK4, CCND1 and P16INK4aGene DNA is copied
Number variation, and analyze the abnormal correlation with patient clinical pathological characters of gene copy number.Wherein, CDK4 expands 173
(40.4%), CDK4 amplifications+Cyclin D1 expand 67 (15.7%), CDK4 amplifications+P16INK4aLack 103 (24.1%).
Carry CDK4 amplifications or P16INK4aIt is normal that the median survival time of the acra type melanoma patients of missing is substantially less than CDK4
Type patient (P=0.041) or P16INK4aNormal type patient (P=0.019).CDK4 amplifications and P16 are carried simultaneouslyINK4aMissing
The median survival time of acra type melanoma patients is substantially less than CDK4 and P16INK4aIt is the patient (P=of normal type
0.003)。
The method used in this experiment is specific as follows：
1.QuantiGene Plex (QGP) method
Gene copy number variation analysis employs the Quantigene Plex experiments based on bDNA technologies.
1. the preparation of homogenate is organized：According to " QuantiGene Sample Processing Kit for FFPE
Tissues (Panomics) " laboratory manuals prepare tissue homogenate.Substantially flow is as follows：5-8 pieces paraffin section is taken to carry out at dewaxing
Reason, then adds 150 μ l Tissue lysates (containing 75 μ g Proteinase Ks), 65 DEG C of incubation 6h.By the postdigestive tissue suspension short time
Centrifugation, abandons precipitation, supernatant is transferred to new pipe.
2. bDNA is tested：Operated according to QuantiGene Plex DNA Assay (Panomics) laboratory manual.Greatly
Body is as follows：With Covaris S2 (Covaris) Ultrasound Instrument, the DNA in homogenate is interrupted with following parametric ultrasonic:Duty
Cycle 5%, Intensity 3, cycles/burst 200,80s.For each experimental port：Take 40 μ l homogenates, plus 5 μ l
2.5M NaOH, 5 μ l DNA probes, 18 μ l Lysis Mixture (are provided) in kit, and the step is carried out at denaturation to DNA
Reason.In this experiment, each sample sets multiple holes to repeat.Terminate after denaturation, reaction solution is transferred to the hybridization plate for completing magnetic bead.
Sealer, in 54 DEG C ± 1 DEG C of shaking table, hybridization reaction 18-22h is carried out with 600rpm revolutions.Next day, in Bio-plex pro
Uncombined probe is washed away in II wash station (Bio-Rad).Then, by magnetic bead in the following order, successively with DNA
Pre-Amplifier, DNA Amplifier, Label Probe and SAPE carry out hybridization reaction.Finally in Bio-plex
Fluorescence signal value is read in 100system (Bio-Rad) enzyme mark working instrument.
3. DNA copy number is calculated：For each sample, first, the mean fluorecence of each gene in two multiple holes is calculated
Value.Then, the Mean Fluorescence of each gene is subtracted into the background value in no specimen blank control, obtains net Mean Fluorescence.
The geometrical mean of tri- reference genes of net Mean Fluorescence divided by RPPH1, RPP30, RPLP0 of testing gene, obtains homogeneous
Change numerical value.For each gene, by the homogenization numerical value of tumor sample divided by the numerical value of standard DNA sample (Affymetrix),
Then 2 are multiplied by, is copy number of the gene in the sample after rounding up.
2nd, real-time fluorescence quantitative PCR (Sonde method) detection P16/Cyclin D1/CDK4 gene copy number changes
Change
1. reaction system (table 1)：
2. reaction condition
Reaction condition：95 DEG C of pre-degenerations 10min, 95 DEG C of denaturation 15s, the simultaneous extension 1min of 60 DEG C of annealing, totally 40 circulations.Often
Individual DNA sample is using 3 multiple holes, and experiment without nucleotides water to replace template as negative control every time, with normal kidney skin stone
The hybrid dna that wax section is extracted is normal reference.
3. P16 is quantitative determinedINK4a, CDK4, CCND1 copy number
WithCopy number assays sonde method real-time quantitative PCRs, ginseng is used as using Rnase P genes
Gene is examined, to target gene P16INK4a/ Cyclin D1/CDK4 are detected that probe article No. is (CDK4:HS02225231_CN；
P16INK4a:HS03717141_CN；CCND1:HS03803699_C N).To eliminate error, made with normal skin tissue's hybrid dna
For normal reference.Reaction is carried out on Applied Biosystems 7500Fast Real-Time PCR System, is used
96 orifice plates, each sample standard deviation is added in 3 multiple holes in triplicate, separately sets at least 2 hole blank controls.PCR reaction systems：20 μ L,
Including：The μ L of Genotyping Master Mix 10, Copy Number Target
Assay 1μL、The μ L of Copy Number Reference Assay 1, DNA profiling n μ L (80ng), double distillations
Water is supplied (8-n μ L).Reaction condition：95 DEG C of pre-degenerations 10min, 95 DEG C of denaturation 15s, 60 DEG C of annealing are simultaneous to extend 1min, totally 40
Circulation.Repeat experiment 1 time.Derived Real-time PCR datas are imported into CopyCaller V2.0 softwares, by normal skin
Tissue DNA sample is set to calibrator sample, and copy number is defaulted as 2.0, calculates the copy number for obtaining tested sample gene
Calculated value and predicted value.
(Δ Δ CT) s, t=μ (Δ CT) s, t- μ (Δ CT) calibrator
RQ (s, t)=2- (Δ Δ CT) s, t
CNsample=RQsample × CNcalibrator
Copy number predicted value for (0,1,2,3+).It is that copy number missing is reduced that copy number, which is less than 2, and copy number is more than 2
Copy number amplification increases.
It is observation terminal with OS (total life span).OS refers to make a definite diagnosis the date to the death time from disease.Through survival analysis,
Carry CDK4 amplifications or P16INK4aIt is normal that the median survival time of the acra type melanoma patients of missing is substantially less than CDK4
Type patient (P=0.041) or P16INK4aNormal type patient (P=0.019).CDK4 amplifications and P16 are carried simultaneouslyINK4aMissing
The median survival time of acra type melanoma patients is substantially less than CDK4 and P16INK4aIt is the patient (P=of normal type
0.003).P<0.05 represents that two groups of difference is statistically significant, and median survival time is the OS obtained after statistical analysis
Median, the middle position Overall survival of patient in representative group.
To totally 514 parts of samples comprising above-mentioned 428 parts of samples (from Beijing Tumour Hospital in January, 2007 to 2015
The clinical samples that October collects, patient's signature informed consent form, voluntarily providing sample is used for this research) carry out above-mentioned experiment to examine
Survey P16INK4a, Cyclin D1, CDK4 gene copy number entire change situations.Wherein, 203 parts show that CDK4 is expanded
(39.5%), 137 parts show that CCND1 expands (26.7%) and 310 parts show P16INK4aLack (60.3%), have
35.2% sample shows comprising two kinds or more of gene copy number variation situation have 8.6% sample to show and include
Three kinds of gene copy number variation situations.For CDK4 amplifications and CCND1 amplifications, further investigations have shown that, include both changes
Most of different sample includes CDK4 the or CCND1 genes of 3-4 copy.
Embodiment two：The effective targeted inhibition agent of cell-based screening
SK-MEL-5 (registration number HTB-70) and A2058 (registration number CRL-11147) cell line purchase used in this experiment
From American Type Tissue Culture (ATCC)；AMC-1~AMC-5 cells come from the clinical samples in embodiment one.
Using primitive cell culture technology, many plants of melanoma primary cell lines are set up, are specially：By in embodiment one
Some patientss specimens from pri is digested to after single cell suspension, is separated except fibroblast obtains tumour cell.Use stem cell
Culture medium in vitro culture and passage, as melanoma primary cell line.Including step：Isolate 1cm3Melanoma sufferer mark
This, is cleaned using ice-cold Dulbecco ' s phosphate buffered solutions (Gibco of ThermoFisher, Waltham, MA).
Tumor tissues are cut into about 1mm3Fragment, and be suspended in 30ml contain 50 × Collagenase IV (Invitrogen) and 1
In the DMEM of × DNA enzymatic (Takara, Kusatsu, Japan), 37 DEG C, 1 hour, to prepare single cell suspension.Suspended described
Liquid is slowly layered on 15ml Histopaque (Sigma, St.Louis, MO), and interface cellular component is collected after rotation.
Then by cell culture is in the serum-free stem cell media of growth factor is supplemented with and passes on, melanoma is obtained primary thin
Born of the same parents system.
Using CellTiter-Glo Luminescent Cell Viability Assay (Promega), to carrying
The acra type melanoma primary cell line of different type CDK4 pathway genes copy number variation has carried out a variety of CDK4 inhibitor
Drug sensitive test.Cell drug sensitivity test is one of the most frequently used experimental technique of translational medicine.Detailed process is as follows：1. black matrix is taken
96 orifice plate bed boards, injection volume is 100 μ l/ holes (2000 cells/wells), culture plate is placed in adherent in incubator；2. cell is treated
Add respective concentration medicine after adherent：15 μ l/ holes, each drug-treated concentration (0.25 μm of ol/L, 0.5 μm of ol/L, 1 μm of ol/L, 2 μ
Mol/L, 4 μm of ol/L) and same concentrations solvent DMSO groups (0.25 μm of ol/L, 0.5 μm of ol/L, 1 μm of ol/L, 2 μm of ol/L, 4 μ
Mol/L three multiple holes) are laid；3. cell ATP enzymatic activity after 24h is detected：Add after CellTilter is incubated 10min and detect extinction
Angle value；4. inhibiting rate is calculated：(control-administration)/control * 100%.
The drug concentration gradient and experimental result that this experiment is used are as shown in Figure 1.Wherein, the medicine used includes LEE011
(#S7440), AT7519 (#S1524) and LY2835219 (#S7158) (be purchased from Sellcek Chemicals (Houston,
TX)), the copy number changes situation contained by each cell in Fig. 1 is as shown in the table：
Table 2：The gene copy number that each cell is included
WT is represented compared with wild type, unchanged.SK-MEL-5 is used as negative control as positive control, A2058.
As seen from the figure, CDK4 pathway inhibitors AT7519 can significantly inhibit CDK4 amplifications+p16INK4aDeletion form cell
AMC-3, CCND1 amplification+p16INK4aDeletion form cell SK-MEL-5 and CDK4 amplification type cell AMC-1 propagation, and to it
Its cell is not acted on significantly；Inhibitor LY2835219 is to the propagation of all cells without obvious inhibitory action；Suppress
Agent LEE011 has inhibitory action when concentration is more than 0.5 μM to AMC-1 cells, does not have obvious inhibitory action to other cells.
Embodiment three：Animal level screens effective targeted inhibition agent
By fresh surgical tissue (patient's acra type melanoma through pathological diagnosis of surgery excision primary or transfer stove)
Transplant in immunodeficient mouse subcutaneously, stable acra type melanoma humanized's Replanting model mice is formed by repeatedly passage
(Patient-derived tumor xenograft, PDTX).Immunodeficient mouse is purchased from the magnificent Fukang biotechnology share in Beijing
Co., Ltd (Institute of Experimental Animals, Chinese Academy of Medical Sciences), mouse age 4-5 week old.Select different type CDK4 path copy numbers
The PDTX models of variation, using CDK4 pathway inhibitor AT7519, observe tumor-bearing mice tumor volume change.Specially：Work as knurl
Body grows to 200mm3When start administration.Experimental group gives CDK4 pathway inhibitors AT7519 and (is purchased from Selleck
Chemicals), physiological saline is dissolved in, is administered (0.3mg/Qd, dosage period 14 days) using intraperitoneal injection mode.Control group with
Same administering mode gives same volume physiological saline.The change of the mouse tumor volume of measurement in every 3 days：V=1/2* minor axis * length
Footpath2, relative volume ratio=V/V0, using the gross tumor volume before being administered for the first time as V0, growth is drawn with Graphpad Prism bent
Line.Wherein, each group includes 4 mouse, every group of relative volume than for 4 mouse each relative volume than average value ± standard
Difference.The gene copy number situation of all types of PDTX models is as shown in table 3 below：
Table 3：Gene copy number situation in PDTX models
Wherein, WT represents unchanged compared with wild type.
As a result as shown in Figures 2 and 3, compared with giving the mouse of same amount of physiological saline, CDK4 pathway inhibitors
AT7519 can make mouse (PDX-001) gross tumor volume of CDK4 amplification+CCND1 amplification type PDTX models substantially be contracted compared with before medication
It is small, can effectively suppress CDK4 amplifications+P16INK4aDeletion form PDTX models (PDX-012), CCND1 amplifications+P16INK4aDeletion form
The growth of the tumour of PDTX model mices (PDX-015), can delay CDK4 amplifications type PDTX model mices (PDX-006) tumour
The speed of growth.For CCND1 amplification type PDTX model mices (PDX-016), using AT7519 groups compared with control group, mouse is swollen
Knurl volume difference is not statistically significant, illustrates that AT7519 may be not suitable for CCND1 amplification type patients.For in the absence of CDK4 paths
The PDTX model mices (PDX-017) of gene copy number variation, growths of the AT7519 to the acra type melanoma be not any
Inhibitory action.
Claims (10)
1. a kind of auxiliary diagnosis and/or the gene marker for treating acra type melanoma, the mark include gene C DK4；
Preferably, in addition to CCND1 or P16INK4a；Or the mark includes gene C CND1 and P16INK4a。
2. gene marker according to claim 1, wherein, described gene C DK4, CCND1 and P16INK4aCopy number deposit
In change.
3. the multiple gene detection kit that a kind of acra type melanoma auxiliary diagnosis and/or medicine for treatment are instructed, the examination
Agent box is used to detect gene marker as claimed in claim 1 or 2；Preferably, the kit is used to detect genetic marker
The copy number of thing.
4. kit according to claim 3, wherein, the kit includes detection gene C DK4, CCND1 and P16INK4a
Probe；Preferably, the probe is respectively CDK4::HS02225231_CN、P16INK4a:HS03717141_CN and CCND1:
HS03803699_CN probes.
5. kit according to claim 3, wherein, the kit includes detection gene C DK4, CCND1 and P16INK4a
Pcr amplification primer thing.
6. gene marker according to claim 1 or 2 the preparation auxiliary diagnosis related to acra type melanoma and/
Or the application in the preparation or kit for the treatment of.
7. a kind of preparation for being used to treat acra type melanoma, the preparation includes CDK4 pathway inhibitors；Preferably, it is described
Acra type melanoma is CDK4 copy number amplifications+P16INK4aThe acra type melanoma of copy number reduction, CDK4 copy numbers expand
The acra type melanoma of increasing+CCND1 copy numbers amplification, CCND1 copy number amplifications+P16INK4aThe acra type of copy number reduction is black
Melanoma or the acra type melanoma of CDK4 copy numbers amplification.
8. preparation according to claim 7, wherein, the CDK4 pathway inhibitors are AT7519, and its structure is shown in formula I
Application of the 9.CDK4 pathway inhibitors in the medicine for preparing treatment acra type melanoma；Preferably, the acra type
Melanoma is CDK4 copy number amplifications+P16INK4aThe acra type melanoma of copy number reduction, the amplification of CDK4 copy numbers+
The acra type melanoma of CCND1 copy numbers amplification, CCND1 copy number amplifications+P16INK4aThe acra type black of copy number reduction
Plain knurl or the acra type melanoma of CDK4 copy numbers amplification.
10. application according to claim 9, wherein, the CDK4 pathway inhibitors are AT7519, its structure such as Formulas I institute
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