临床病理学-肿瘤病理的分子检测

肿瘤病理的分子检测郑州大学一附院病理科分子病理常用检测技术Taqman-ARMS（与国际获批的技术相当）DNA测序（基因突变检测的金标准技术）NGSFISHSchematic

representation

of

the

PI3K-AKT

(AKT)

signaling

pathway

(57).

Anextracellular

factor

such

as

a

growth

factor

interacts

with

itsreceptor

proteintyrosine

kinase

(RPTK)

resulting

in

autophosphorylation

oftyrosine

residues.

Phosphatidylinositol-3

kinase

(PI3K)

consisting

ofan

adaptor

subunit

p85

and

a

catalytic

subunit

p110

is

translocated

to

the

cell

membrane

and

binds

to

phosphotyrosine

consensus

residues

of

theRPTK

through

ist

its

adaptor

subunit.

This

results

in

allosteric

activation

of

the

catalytic

subunit

leadingto

production

of

phosphatidylinositol-3,4,5-triphosphate

(PIP3).

PIP3

recruits

signaling

proteins

with

pleckstrin

homolgy

(PH)

domains

to

the

cell

membrane

including

AKT.

PTEN(phosphatase

and

tensin

homologue

deleted

fromchromosome

10)

is

a

PIP3

phosphatase

and

negatively

regulates

the

PI3K-AKT

pathway.

Theinteraction

of

PIP3

withthe

PH

domain

of

AKT

likelyinduces

conformational

changes

in

AKT,

therebyexposing

the

two

main

phosphorylationsites

at

T308

and

S473.

T308

and

S473

phosphorylation

by

protein

serine/threonine

kinase

3"-phosphoinositide-dependent

kinases

1

and

2(PDK1

and

PDK2)

is

required

for

maximal

AKTactivation.

Activated

AKT

translocates

to

the

nucleus

and

mediates

the

activation

and

inhibitionof

various

targets

resulting

in

cellular

survival

and

cell

growth

and

proliferation.PTK=protein

tyrosinekinase相关的信号通路----PI3K-AKT(AKT)信号通路Schematic

representation

of

the

Ras-Raf-MEK-ERK

(MAPK)

signaling

pathway.

Anextracellular

factor

such

as

a

growth

factor

(GF)

interactswith

its

receptor

tyrosine

kinase

(RTK)

and

induces

receptor

dimerisation

and

autophosphorylation

on

tyrosine

residues.

The

phosphotyrosinesfunctionas

dockingsites

for

the

growth-factor-receptor-bound

protein

2

adapter

protein

(Grb2).

Grb2

pulls

the

GDP/GTP

exchange

factor

sonof

sevenless

(SOS)

to

the

cell

membrane.

SOS

induces

switching

of

the

Ras-family

GTPases

from

the

inactive

GDP-bound

state

to

the

activeGTP-bound

state.

Activated

Ras

binds

to

the

Raf

serine/threonine

kinases

(A-Raf,

B-Raf,

C-Raf/Raf-1)

and

recruits

themto

the

cell

membrane.Activation

of

B-Raf

is

obtained

after

binding

to

Ras

alone

whereas

for

activation

of

A-Raf

and

Raf-1

additional

signals

are

required.

Raf-1activation

isa

multi-step

process

that

requires

the

phosphorylation

of

activating

sites

by

other

kinases

(e.g.

Src)

as

well

as

the

dephosphorylationof

inhibitory

sites

by

protein

phosphatase

2A

(not

shown).

Activated

Raf

phosphorylates

and

activates

MEK

which

phosphorylates

and

activatesERK.

The

Raf-MEK-ERK

cascade

is

scaffolded

by

the

kinase

suppressor

of

Ras

(not

shown).

Activated

ERK

has

many

substrates

in

the

cytosol

and

can

also

enter

the

nucleus

to

regulate

gene

expression

by

phosphorylating

transcription

factors

(TFs).相关的信号通路----Ras-Raf-MEK-ERK(MAPK)信号通路EGFR

historyJCO2020303标志小分子靶向药物治疗时代到来Prof.

Richard

of

President

of

ASCOpointed

out

that

“We

now

need

to

thinkabout

NSCLC

as

at

least

2

distincttypes

of

cancer”.Frequency

ofmutations

inexons

18–21

of

the

EGFR

gene

and

the

association

withresponsiveness

to

EGFR

targeted

therapy.The

EGFR

located

in

chromosome

7p11.2

contains

28

exons.

Exons

18–21

in

the

tyrosine

kinase

region

of

the

EGFR

gene

are

scaled

up;

adetailed

list

of

EGFR

mutations

in

these

exons

associated

with

sensitivity

(green)

or

resistance

(orange)

to

EGFR

TKI.6,12,67–71,80–84,195Thefrequency

of

the

mutations

is

labeled

to

the

side

of

the

color-coded

bars.

The

most

prevalent

EGFR

mutations

are

in-frame

deletions

of

exon

19

(45%),

followed

by

L858Rsubstitution

in

exon

21

(41%).

Exon

18

mutations

(G719A/C/S)

account

for

B5%of

the

overallmutations.

The

exon

19

deletions,

L858R

in

exon

21,

G719A/C/S

in

exon

18,

the

L861Q

and

L861R

in

exon

21,

are

mutations

that

predictthe

probability

of

benefit

from

EGFR

TKI

therapy

of

adenocarcinomas.

The

insertion

mutations

in

exon

20

(D770_N771

(insNPG),D770_N771

(insSVQ),

D770_N771

(insG))

are

the

second

most

common

and

are

associated

withEGFRTKI

therapy

resistance.

D761Y

inexon

19

is

also

associated

withresistance

to

EGFRTKI

although

it

occurs

in

low

frequency.

*T790M

mutation

represents

B1%

of

primary

resistance

but

over

50%of

acquired

resistance

in

adenocarcinomas.

**There

are

more

than

20

exon

19

deletion

forms

in

the

lungadenocarcinomas,

with

the

most

common

ones

including

delE746-A750,

delL747-T751linsS,

and

delL747–P753insS.EGFR基因突变位点靶向药物：易瑞沙，特罗凯检测：定量PCR或测序。耐药突变敏感突变八项随机研究奠定了EGFR-TKI在EGFR突变阳性患者中的一线治疗地位研究N(EGFREGFR突变类型ORR

(%)PFS(月)HR

PFSm+)IPASS26119Del/L858R

+

other

(8%)71.2

vs

47.39.8

vs

6.40.48First-SIGNAL4219Del/L858R84.6

vs

37.58.4

vs

6.70.61WJTOG

340517219Del/L858R62.1

vs

32.29.6

vs

6.60.49NEJGSG00222419Del/L858R

+

other

(6%)73.7

vs

30.710.8

vs

5.40.30OPTIMAL15419Del/L858R83

vs

3613.1

vs

4.60.16EURTAC17319Del/L858R58

vs

159.7

vs

5.20.37LUX-LUNG

330819Del/L858R

+

other

(11%)61

vs

2211.1

vs

6.90.58LUX-LUNG

636419Del/L858R

+

other66.9

vs

23.011.0

vs

5.60.28Mok

et

al

NEJM

2009,

Lee

et

al

WCLC

2009,

Mitsudomi

et

al

Lancet

Oncology

2010,Maemondo

NEJM

2010,

Zhou

et

al

ESMO

2010,

Rosell

Lancet

Oncol

2012,Yang

JC

et

al

ASCO

2012,

Wu

YL

et

al

ASCO

2013易瑞沙，特罗凯，凯米娜Biochemical

pathways

leading

to

resistance

to

small-molecule

epidermal

growth

factor

receptor

(EGFR)-targeting

drugs

such

as

gefitinib

anderlotinib

innon-small-cell

lung

cancer

(NSCLC).

Simplified

pathwaydiagram

showing

EGFR

signaling

through

the

RAS/MEK/ERK

andPI3K/PDK1/AKT

pathways,illustrating

the

points

of

mutation/amplification

in

EGFR

TKI

resistance,

along

with

other

mechanisms.

The

resistance

mechanisms

include

theEGFR

p.T790Mgatekeeper

mutation,

amplification

of

EGFRp.T790M,

MET

amplification,

PI3KCAmutation,

and

an

at

least

two-fold

increase

in

the

expressionof

GAS6

and

itsreceptor

AXL.

Incidence

rates

are

givenwhere

known.

The

FAS/nuclear

factor-κB

(NF-κB)

signaling

armdownstreamof

the

FAS

deathreceptor

has

also

beenshown

to

be

important

in

EGFR

tyrosine

kinase

inhibitor

resistance.

In

addition,

epithelial-to-mesenchymal

(EMT)

transitionchanges,

perhapsassociatedwithincreased

activity

of

AXL,

and

transformation

from

the

NSCLC

to

the

small-celllung

cancer

(SCLC)

phenotype

can

lead

to

decreasedresponsiveness.

Theidentification

of

various

resistance

mechanisms

suggests

that

a

range

of

clinically

actionable

therapies

could

be

used

to

overcome

the

resistance.Formoredetails,

see

text.EGFR-TKI获得性耐药两个主要原因

MET扩增：克唑替尼，

FISH

T790M二次突变：

AZD9291,PCR或测序Selumetinib

司美替尼肺腺癌：基因指导的个体化治疗——药靶图凡德他尼阿法替尼吉非替尼厄洛替尼阿法替尼克唑替尼瑞戈非尼？MEK抑制剂舒尼替尼克唑替尼针对肺腺癌的靶向药物肺腺癌15–25%存在KRAS突变，肺鳞癌罕见。KRAS基因突变KRAS突变在预测E-TKIs治疗效果或预后方面的作用并不一致。G12C/G12V突变亚组的预后更好但E-TKIs治疗效果更差，G12D/G12S突变亚组预后较差，但可从E-TKIs治疗中获益。这些观察结果还有待进一步验证。2014

ASCO检测：定量PCR或测序。pproximately

3–7%

of

lungtumors

harbor

ALK

fusions

(Koivunen

etal.

2008;

Kwak

etal.

2010;

Shinmura

etal.

2008;

Soda

etal.

2007;Takeuchi

et

al.

2008;

Wong

et

al.

2009).

ALK

fusions

are

more

commonly

found

in

light

smokers

(<

10

pack

years)

and/or

never-smokers(Inamura

etal.

2009;

Koivunen

etal.

2008;

Kwak

et

al.

2010;

Soda

etal.

2007;

Wonget

al.

2009).

ALK

fusions

are

also

associated

withyounger

age

(Inamura

etal.

2009;

Kwak

et

al.

2010;

Wong

et

al.

2009)

and

adenocarcinomas

with

acinar

histology(Inamura

et

al.

2009;

Wongetal.

2009)

or

signet-ring

cells

(Kwak

et

al.

2010).

Clinically,

the

presence

of

EML4-ALK

fusions

is

associated

with

EGFRtyrosine

kinaseinhibitor

(TKI)

resistance

(Shawet

al.

2009).Multiple

different

ALK

rearrangements

have

been

described

in

NSCLC.

The

majority

of

these

ALK

fusionvariants

are

comprised

of

portions

ofthe

echinodermmicrotubule-associated

protein-like

4

(EML4)

gene

with

the

ALK

gene.

At

least

nine

different

EML4-ALK

fusion

variants

havebeen

identified

in

NSCLC

(Figure

1;

Choi

etal.

2008;

Horn

and

Pao

2009;

Koivunen

etal.

2008;

Soda

etal.

2007;

Takeuchi

et

al.

2008;Takeuchi

et

al.

2009;

Wong

et

al.

2009).

In

addition,

non-EML4

fusionpartners

have

also

been

identified,

including

KIF5B-ALK

(Takeuchi

et

al.

2009)

and

TFG-ALK

(Rikova

etal.

2007).

Clinically,

the

presence

of

an

ALK

rearrangement

is

detected

by

fluorescence

in

situ

hybridization(FISH)

with

an

ALK

break

apart

probe.

FISH

testing

is

not

able

to

discern

which

particular

ALK

fusion

is

found

in

aclinical

sample.In

the

vast

majority

of

cases,

ALK

rearrangements

are

non-overlappingwith

other

oncogenic

mutations

found

inNSCLC

(e.g.,

EGFR

mutations,KRAS

mutations,

etc.)

(Inamura

etal.

2009;

Kwak

etal.

2010;

Shinmura

et

al.

2008;

Wongetal.

2009)..ALK基因重排在NSCLC中…发生率： 3-7%临床特点：少吸（<10包、年）/不吸烟年轻患者腺泡或印戒细胞癌融合特点：主要与EML4存在至少9种融合方式，其他IFG-ALK,

KIF5B-ALK与其他癌基因变异不共存靶向药物：克唑替尼临床检测方法：FISH，增强免疫组化，RT-PCRROS1

is

a

receptor

tyrosine

kinase

(RTK)

of

the

insulin

receptor

family.

Chromosomal

rearrangements

involving

the

ROS1

gene,onchromosome

6q22,

were

originally

described

in

glioblastomas

(e.g.,;

Birchmeier,

Sharma,

and

Wigler

1987;

Birchmeier

et

al.

1990;

Charest

etal.

2003).

More

recently,

ROS1

fusions

were

identified

asa

potential

"driver"

mutation

in

non-small

cell

lung

cancer

(Rikova

et

al.

2007)

andcholangiocarcinoma

(Gu

et

al.

2011

).ROS1

fusions

contain

an

intact

tyrosine

kinase

domain.

To

date,

those

tested

biologically

possess

oncogenic

activity

(Charest

et

al.

2003;

Rikovaet

al.

2007).

Signaling

downstreamof

ROS1

fusions

results

in

activation

of

cellular

pathways

knownto

be

involved

in

cell

growth

and

cellproliferation(Figure

1).

ROS1

fusions

are

associated

with

sensitivity

invitro

to

tyrosine

kinase

inhibitors

that

inhibit

ROS1

(McDermott

etal.

2008).Schematic

representation

of

ROS1

fusions.

"X"

represents

the

various

fusion

partners

that

have

been

described.

Dimerization

of

the

ROS1

fusionmediated

by

the

fusion

partner

("X"),

results

in

constitutive

activation

of

the

ROS1

tyrosine

kinase.

ROS1

signaling

results

in

pro-growth

and

anti-apoptosis

effects.ROS1、RET基因重排ROS1重排见于2%肺肿瘤；少吸（<10包、年）/不吸烟患者；年轻患者；腺癌。临床对克唑替尼敏感。对EGFR

TKIs不敏感。RET基因融合见于1.3%肺癌，腺癌。临床检测方法：FISH，RT-PCRHER2在乳腺癌中…HER2在乳腺癌中…HER2扩增与肿瘤发生有关。肿瘤体积大,无病生存期短,对CMF等方案耐药,对蒽环类药物比较敏感,50%患者为ER或PR阳性。乳腺癌18–20%呈HER2过表达。过表达主要源于基因扩增。应用FISH检测。靶向药物Heceptin仅对HER2扩增的乳腺癌有效准确的检测HER2有无扩增是临床应用Heceptin的绝对必要条件，也是成功进行靶向治疗的前题和关键胃癌HER2阳性率10%～38%胃癌EGFR表达阳性率42%-77.1%（IHC）EGFR基因热点位点突变罕见：0%-2.6%Kras突变率0-10%Braf突变率0-2.3%PIK3CA突变率6%胃癌基因表达和相关基因的突变胃癌HER2异质性表达：部位异质性：胃食管结合部癌(32%)高于胃体(20.9%)类型异质性：肠型(32.1%)胃癌高于弥漫型胃癌(6%)肿瘤组织内异质性MSI/MMR：肠癌预后及5FU疗效预测微卫星是一种短串联重复序列即DNA重复序列，以1～6个核苷酸为一个单位重复10～60次。

MSI

（microsatellite

instability，微卫星不稳定性）：由于重复单位的插入或缺失

而造成的微卫星长度的任何改变，出现新的微卫星等位基因。MSI由MMR

（mismatch

repair，错配修复基因）缺陷造成。MMR基因（主要是MLH1、MSH2、PMS2、MSH6）失去功能，导致不能修复DNA复制过程中出现的错配，进而产生MSI表型。MSI-H（高度微卫星不稳定）见于90%的林奇综合征和10%~15%的散发性结直肠癌11.

Expert

Rev

Gastroenterol

Hepatol,2011,5(3):385-399.MSI结直肠癌的病理特征(散发性结直肠癌)①散发性结直肠癌多位于近端结肠，易伴发肠内或肠外其他器官的多发性肿瘤②大约15％的散发性结直肠癌显示MsI③较少发生淋巴结转移，生物学行为较好④与MSS比较，MSI肿瘤复发率低⑤肿瘤细胞DNA多为二倍体或近二倍体⑥其病因通常是hMLHl基因启动子甲基化，MMR基因沉默导致免疫组化显示相关MMR蛋白阴性，表现MSI⑦对某些化疗药物(如5Fu、顺铂等)有原发性耐药⑧免疫组化检测的灵敏度与特异度分别为83%和88.8%，而

MSI检测为89%和90%PRIME（2013）：从KRAS到RASOliner,

et

al.

ASCO

2013;

Schwartzberg,

et

al.

ASCO

2013Douillard,

et

al.

NEJM

2013Panitumumab

+

FOLFOX4

(n=593)(n=325

KRAS

WT

(exon

2);n=320

in

KRAS/NRAS

analysis)FOLFOX4

(n=590)(n=331

KRAS

WT

(exon

2);n=321

in

KRAS/NRAS

analysis)RPreviously

untreated

mCRC(n=1,183)(n=641

in

KRAS/NRAS

analysis)KRAS

geneExon

1Exon

2Exon

3Exon

4NRAS

geneExon

1Exon

2Exon

3Exon

44%6–7%0%4–6%3–5%40%

基于PRIME研究Biomarker分析结果，EMA（欧洲药监局）于2013年6月27日发布帕尼单抗适应症修改信息：仅用于RAS野生型患者

基于OPUS研究Biomarker分析结果，EMA于2013年11月21日发布西妥昔单抗适应症修改信息：仅用于RAS野生型患者

规定对RAS突变状态的检测应包括KRAS(exons2,3,and

4)和NRAS(exons

2,3,and

4)Biomarker检测：推动治疗方案发展BRAF：突变预后更差CRYSTAL/OPUS综合分析提示：BRAF突变是预后不良因素之一，但不能预测西妥昔单抗疗效1

。0

6

121.

Eur

J

Cancer.

2012

Jul;48(10):1466-750

.40

.30

.20

.10

.0OS

estima0t.t.5e0

.60

.70

.80

.91

.0182460303642KRAS野生/BRAF突变HR

0.62

(95%

CI

0.36–1.06);

p=0.076CT

+

cetuximab

(n=32):

mOS

14.1

moCT

(n=38):

mOS

9.9

mo48

54时间(months)KRAS野生/BRAF野生HR

0.84

(95%

CI

0.71–1.00);

p=0.048CT

+

cetuximab

(n=349):

mOS

24.8

moCT

(n=381):

mOS

21.1

moUGT1A

基因簇,共包含17个外显子(图1),共用2～5外显子,编码UGT1A蛋白共同C末端,是尿苷二磷酸葡萄糖醛酸(UDPGA)结合域,共有246个氨基酸残端,组成了UGT1A的保守序列;其余13个外显子,均为第1外显子,序列有37%～90%的同源性,编码UGT1A蛋白特异的N端,有285个氨基酸残基,是UGT1A的底物识别区。13个第1外显子共编码9种功能性UGT1A

蛋白(UGT1A1、UGT1A3、UGT1A4、UGT1A5、UGT1A6、UGT1A7、UGT1A8、UGT1A9、UGT1A10)和4个假基因(UGT1A12P、UGT1A11P、UGT1A13P、UGT1A2P)UGT1A1是一种什么酶尿苷二磷酸葡糖醛酰转移酶（UGTs)是一大类能催化葡萄糖醛酸与亲核底物结合的膜结合酶，主要存在于肝脏人类的UGTs被分为UGT1，UGT2两个家族UGT1的基因至少包括13个亚型：UGT1A1，UGT1A3-10，UGT1A12P、UGT1A11P、UGT1A13P、UGT1A2PUGT1A1，降解伊立替康。A（腺嘌呤）、T（胸腺嘧啶）、G（鸟嘌呤）、C（胞嘧啶UGT1A1UGT1A1

*27686

C>AUGT1A1

*291099

A>CUGT1A1

*6211G>AUGT1A1

G-3156AUGT1A1

*1 A(TA)6

TAAUGT1A1

*28 A(TA)7

TAAUGT1A1

*33 A(TA)5

TAAUGT1A1

*34 A(TA)8

TAA启动子区EXONGenet

Med

2009:11(1):21–34不同人群的发生基因型或突变概率略有不同！c-kit/PDGFRA突变类型预测伊马替尼疗效，其中c-kit外显子11突变疗效最佳PDGFRA

D842V突变者对伊马替尼原发耐药。检测方法：DNA测序胃肠道间质瘤与格列卫基因重排基因重排基因重排对淋巴瘤诊断的意义确定淋巴组织增生性疾病的克隆性 区分淋巴瘤和反应性增生区别肿瘤性B和T细胞检查微小残余灶确定淋巴瘤细胞的来源几乎所有的B细胞淋巴瘤表现出Ig重链和轻链基因单克隆性重排大多数T细胞淋巴瘤显示TCR基因单克隆性重排淋巴瘤相关分子诊断BCL6基因断裂检测意义（双色分离探针）BCL6基因重排的检测可以辅助诊断弥漫性大B细胞淋巴瘤。BCL6阳性患者诊断治疗36个月后，疾病停止发展的比率为82%，携带有BCL6重排的病例预后较好。C-MYC基因断裂检测意义（双色分离探针）约80%的伯基特淋巴瘤病例发生t(8；14)（q24;q32）；约15%的伯基特淋巴瘤病例发生t(2；8)(p11;q24)；约5%发生t(8；22)（q24;q11）。C-MYC基因断裂重组可能是伯基特淋巴瘤的一个标志，可以应用于临床上伯基特淋巴瘤的辅助诊断BCL2/IGH融合基因检测意义（双色双融合探针）辅助诊断滤泡性淋巴瘤BCL2/IGH持续阴性的FL患者3年生存率为100%，而阳性者3年生存率只有54