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

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。

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

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

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

“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

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

Indirect ( repressing tumor suppressor gene expression); direct (promotes mutations )

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

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

肿瘤是难治的重大疾病之首

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

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

　 　 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

1. Before 2, After

A sensitive/timely assessing the quality of the surgery;

15In confidence

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

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

+

-

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!!

The circulating DNA

in plasma

Standard

Cell free DNA

Run through(Other

components in plasma)

Salt

19

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

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).

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

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

Eight chemo-therapeutics

//Eight bladder cancer

Cell lines and oneImmobilized normal cell line

Eight chemo-therapeutics

//Eight bladder cancer

Cell lines and oneImmortalized normal cell line

Cancer cell lines

An immortalized benigncell line

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

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

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

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)

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

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

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