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A systems approach to the improved control of cancer threat:
A bladder cancer paradigm
Jingde Zhu
Collaborators Bladder cancer
C. Zhang, 哈医大一附院J. Xiao , Anhui 省立医院
Anhui Cancer HospitalHui Deng
Lei LvYang Li
Ran XiaHuanhuan Zhang
Sanqiang NiuYu LiangXun Chen
Xiaohui HanHuanhuan Zhang
Fengyu Zhu
Shanghai Cancer InstituteHongyu Zhang
Yinghua He
http://www.bio360.net/z/Cancer/index.html
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1, 早诊无门 ;(尿沉淀的 DNA 甲基化分析和
膀胱癌的早期诊断)
2, 治疗盲目无效 ; (尿沉淀的DNA甲基化分析和
膀胱癌的理想化疗)
3, 缺乏有效的治疗方案(表型驱使算法指导的组合化疗
方案的快速优化)
肿瘤是难治的重大疾病之首
肿瘤是唯一的以异常增殖为特征,克服了肌体维稳机制的复杂重大疾病。具有极为不稳定的基因组,
表现为高度适应性和在从遗传,表观遗传和多个层面的高度异质性。
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Ignorance is a source of all evils
Coming Full Circle—From Endless Complexity to Simplicity and Back
AgainWeinburg, R, Cell 2014 March
1980 2000
Descriptive Reductionist
Massive dataLittle knowledge
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The precision medicine, the “ mission impossible ?”1, A huge gap between What we can define
versus what we can interpret
2, Other causes :A, Prior-: Poor designs and sampling bias ;B, During: Technological error ;C, Post-analysis :Including the mis-interpretaion of the phenotypic impact of the definable features
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Mutation, Expression ( protein and mRNA/miR)
Cancer biomarker Sensitive, Specific,
Cost-effective, Fast,
Robust against inter-operator and inter-institutional variability,Better than the existing diagnostics.
The promising // yet extensively explored
5In confidence
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“Guidelines for the Use of Tumor Markers in Bladder Cancer”By National Academy of Clinical Biochemistry(NACB)
Not recommended for 1, screening or diagnosis of bladder tumors.
2, monitoring patients after treatment for bladder cancer.
Genomic Amplification
Protein expression
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Genotype
Epigenotype
Stored and Inert
(Organized and functional)
PhenotypeMultiple
dimensional
Transcriptionaland post-transcriptional
Environment
Exp
ression
p
rofile
One dimensional
4-dimensional
Flexible and dynamic
Epigenomics comes of age; The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence
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Indirect ( repressing tumor suppressor gene expression); direct (promotes mutations )
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Transcriptionaland post-transcriptionalE
xpressio
n
pro
fileGenotype
一维, 存储的
Epigenotype
( 功能性,多维, 可塑 ) 多维,多变表型
1, 遗传性 ;
2, 功能性 和表性效应
表型效应 (Y axis)
稳定性 (Y axis)决定肿瘤行为核心基因群 》 受到DNA甲基化调控的
DNA methylation is the most promising type of cancer biomarkers
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DNA methylation cancer diagnostics for early detection of
bladder cancer
( multi-omic analyses of the normal versus cancer cell lines
)
( Clinical validation)
(the multiplex q-PCR assay)
Brickman, etal., 2010, MethodsXiong, et al, 2010, Nature BiotechLi, et al, 2010, Plos BiolLi et al, 2012, BMC genomics
Yui et al, 2007 Clinic Cancer Res, Ma, e tal., 2012, JBCCao, et al, 2013, Cancer ResLv, et al, 2014, Cell Death and DiseaseDeng, et al, 2014, Molecular Cancer Lv, et al 2015, Cancer Letter ,Deng et al 2015 , BBA Disease Li , et al 2015 , Onotarget
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肿瘤是难治的重大疾病之首
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The gold standard diagnosis
Invasive
Subjective
Misdiagnosis: 10-40%Urine cytology(Specific but insensitive)
A, DNA methylation analysis in urine sediment for the early detection of bladder cancer
Low sensitivity
Bladder cancer is a costing and difficult
type of disease to be contained.
1, Often occur in multi-lesions2, Recur frequently3, Chemoresistant
Sample : Cells from 50 ml morning Urine
For DNA methylation analysis
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Methylomic profiling of two cases of the normal tissues and two bladder cancer cell lines
1 , the cancer methylomic profile;2, the candidates for cancer detection;
Methylomic profiling technology
2, MBD affinity
chromatography
1, Sonicated DNA/Adaptor(1)
Sequencing
Unbound (2)
(3)
(4)Elution
Several thousands of the cancer statespecific hypermethylated targets
The diagnostic kit
An informative set of 5
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1 2 3 4 5 Bladder cancer (131) 65.6% 34.4% 51.1% 48.1% 67.2%
Non cancerous lesion (36)
0.00% 0.00% 11.11% 11.11% 13.89%
Accumulated sensitivity 65.6% 71.0% 80.5% 88.5% 94.75Accumulated specificity 100% 100% 88.9% 86.1% 83.3%
71%
94.75%
100% (11/131 cases )detection rate in the veryearly stage of disease.
14In confidence
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1. Before 2, After
A sensitive/timely assessing the quality of the surgery;
15In confidence
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1, Sample extraction
Kit
Fam(480nm) HEX(520nm) Rox(580nm) Cy5(645nm)
Target 1 Uncut control Target 2 Cut control
Set 1,
Fam(480nm) HEX(520nm) Rox(580nm) Cy5(645nm)
Target 3 Uncut control Target 4 Target 5
Set 2,
2,Methylation sensitive restriction
--CG--- --mCG---
4, multiplex
3,qPCR
The methylation sensitive digestion/4-plex qPCR assay
X 2Five targets
One digestion controlPlus one undigetstion control
The methylation sensitive digestion/4-plex qPCR assay
X 2Five targets
One digestion controlPlus one undigetstion control
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In confidence, Jingde Zhu, 2011 0809
17
Target/the internal control(cut)
Target/the internal control(uncut)
Y axis: (The difference in the cycle of PCR)
///////Cancer Cell
Normal Tissue
100% 20ng 0ng
5 % 1ng 19ng
0.5% 0.1 20 ng
0% 0ng 20ng
Cot (cut-uncut)
Fam(480nm)
HEX(520nm)
Rox(580nm)
Cy5(645nm)
Target 3
Uncut control
Target 4
Target 5
+
-
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CTC: Circulating Tumor Cells
Cancer cells(very few)remains in circulation
Metastasis
Survival
Seeding and invading
Proliferating
Cancer Cells (Vast majority) lyse and their DNA remains in
circulation for various lengths of the time.
Circulating DNA
Normal ; <5ng/ ml plasmaCancer: >> 10ng/ ml
Cancer specificchanges!!
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The circulating DNA
in plasma
Standard
Cell free DNA
Run through(Other
components in plasma)
Salt
19
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As much as 5 ng DNA can be recovered from 1 ml serum (or plasma)
Input Recovered
Input Recovered
Conclusion : As little as 0.2ng DNA is MSPable.
Treaded template
1 2 3 4 —
20ng
5ng 1 ng 0.2 ng —
A robust MSP of 0.2 ng amount of template
20
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The cancer heterogeneity reflects the mismatch between cost and
benefit of some anticancer therapies (Fojo et al., 2014)
Between 2002 and 2012, 71 anticancer drugs (52 targeted drugs)approved
the median overall survival benefit was 2.1 months ( costing estimated $10,000 per month on therapy 等于 $2.7 million per life year saved (Kantarjian and Zwelling, 2013).
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1, 早诊无门 ;(尿沉淀的 DNA 甲基化分析和
膀胱癌的早期诊断)
2, 治疗盲目无效 ; (尿沉淀的DNA甲基化分析和
膀胱癌的理想化疗)
3, 缺乏有效的治疗方案(表型驱使算法指导的组合化疗
方案的快速优化)
肿瘤是难治的重大疾病之首
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B, For DNA methylation diagnosis for the guided chemotherapy of bladder cancer
SNPs/INDELs
Copy number variation
Other structure variation
mRNA expression
lncRNA expression
Alternative splicing
DNA methylation
Exo
me-seq
RN
A-seq
Cell lines of distinct chemoresistance
IC50
DF genes ( pathways)
Functional annotationBioinformatically and
experimentally
GO/KEGG/IPA
( 9 cell lines/8 drugs) (mono, bi- or tri- omic
analyses)1, For better mechanistic understanding
2, For better DNA methylation diagnotics
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Eight chemo-therapeutics
//Eight bladder cancer
Cell lines and oneImmobilized normal cell line
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Eight chemo-therapeutics
//Eight bladder cancer
Cell lines and oneImmortalized normal cell line
Cancer cell lines
An immortalized benigncell line
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The miR-193a-3p as the chemoresistant gene
DNA methylation ( 1, MBD-capture seq)
Transcription repression
Expression of Protein coding (2,mRNA seq)
Expression of miR gene (3, miR seq)
Post-transcriptional repression
the tri-omic analyses
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H-bc 5637
Chemo-resistant High Low
miR-193a expression High Low
DNA methylation state Low High
The chemo-resistance associated DF expression
/methylation of the miR-193a gene
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IC50
Profiling
BCa cellline
s
5637
H-bc
MiR-omic
Methylomic
miR-193a
RNA-seq
Predicted and
reported targets
candidatesTarget genes
True Targets
Cell chemo-sensitivity
TumorXenograf
t
Pathway
Reporter Array
Pathways related
Forced reversal expression
validation
validation
Knock down
Chemo-sensitive
Chemo-resistant
Pa, Pi, Ad, EH
Pa injectio
n
T24
Biu87
EJ
VS
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Targets of miR-193a-3pmiR-193a-3p
Coding genes Noncoding genes
SRSF2 HIC2 PLAU
PSEN1 LOXL4 ING5
HOXC9
Lnc-AKIP1-
3
Lnc-DHX38-
5Other
LncRNAs
Lv et al, Cell Death and Disease 2014; Deng, et al, Molecular Cancer 2014;Li et al Oncotarget ( revised), Lv et al Cancer Letter (revised)Deng, et al, BBA disease ( revised)
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miR-193a-
3p
seven identifi
ed target
sOther target
s
(-)(-)
(+)Chemo-
resistance & Cell
survival
DNA hypermethyla
tion
Pa Ad
EH PiCi
Pa, Pi, EH , Ad ,Pa, Ad Pa, Ad
Pa, Pi, Ci, EH,
Pi, Ci, EH, Ad
Ci, PiCi, Pi,
EH
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Comprehensive profiling
In confidence 32
Biomarkers Analysis CollaboratorMutation PCR or seq 医科院肿瘤医院 Dr. Jiao
DNA methylation Methylation disgestion/qPCR
Urigenital tract microbiome
Seq 华中科技大学, Dr. Ning, Dr. Han
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Normalhepatocyte
Disease
Hepatocellularcarcinoma
1, Purification of 1) hepatocytes ( or 2) their
precursors)
1)
2)
1) Amplification in cell culture
2, Sample precessing for mapping of 1), DNA methylation; 2), transcription factors;
And 3), histone modification
3, Data retrieving, analysis, integration and sharing
1), Array, and 2), Deep sequencing
The Chinese Hepatocyte Epigenome Program Proposed