臨床病理學(xué)-腫瘤病理的分子檢測(cè)

腫瘤病理的分子檢測(cè)鄭州大學(xué)一附院病理科分子病理常用檢測(cè)技術(shù)Taqman-ARMS（與國(guó)際獲批的技術(shù)相當(dāng)）DNA測(cè)序（基因突變檢測(cè)的金標(biāo)準(zhǔn)技術(shù)）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相關(guān)的信號(hào)通路----PI3K-AKT(AKT)信號(hào)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).相關(guān)的信號(hào)通路----Ras-Raf-MEK-ERK(MAPK)信號(hào)通路EGFR

historyJCO2020303標(biāo)志小分子靶向藥物治療時(shí)代到來(lái)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基因突變位點(diǎn)靶向藥物：易瑞沙，特羅凱檢測(cè)：定量PCR或測(cè)序。耐藥突變敏感突變八項(xiàng)隨機(jī)研究奠定了EGFR-TKI在EGFR突變陽(yáng)性患者中的一線治療地位研究N(EGFREGFR突變類型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，特羅凱，凱米娜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獲得性耐藥兩個(gè)主要原因

MET擴(kuò)增：克唑替尼，

FISH

T790M二次突變：

AZD9291,PCR或測(cè)序Selumetinib

司美替尼肺腺癌：基因指導(dǎo)的個(gè)體化治療——藥靶圖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抑制劑舒尼替尼克唑替尼針對(duì)肺腺癌的靶向藥物肺腺癌15–25%存在KRAS突變，肺鱗癌罕見(jiàn)。KRAS基因突變KRAS突變?cè)陬A(yù)測(cè)E-TKIs治療效果或預(yù)后方面的作用并不一致。G12C/G12V突變亞組的預(yù)后更好但E-TKIs治療效果更差，G12D/G12S突變亞組預(yù)后較差，但可從E-TKIs治療中獲益。這些觀察結(jié)果還有待進(jìn)一步驗(yàn)證。2014

ASCO檢測(cè)：定量PCR或測(cè)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…發(fā)生率： 3-7%臨床特點(diǎn)：少吸（<10包、年）/不吸煙年輕患者腺泡或印戒細(xì)胞癌融合特點(diǎn)：主要與EML4存在至少9種融合方式，其他IFG-ALK,

KIF5B-ALK與其他癌基因變異不共存靶向藥物：克唑替尼臨床檢測(cè)方法：FISH，增強(qiáng)免疫組化，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重排見(jiàn)于2%肺腫瘤；少吸（<10包、年）/不吸煙患者；年輕患者；腺癌。臨床對(duì)克唑替尼敏感。對(duì)EGFR

TKIs不敏感。RET基因融合見(jiàn)于1.3%肺癌，腺癌。臨床檢測(cè)方法：FISH，RT-PCRHER2在乳腺癌中…HER2在乳腺癌中…HER2擴(kuò)增與腫瘤發(fā)生有關(guān)。腫瘤體積大,無(wú)病生存期短,對(duì)CMF等方案耐藥,對(duì)蒽環(huán)類藥物比較敏感,50%患者為ER或PR陽(yáng)性。乳腺癌18–20%呈HER2過(guò)表達(dá)。過(guò)表達(dá)主要源于基因擴(kuò)增。應(yīng)用FISH檢測(cè)。靶向藥物Heceptin僅對(duì)HER2擴(kuò)增的乳腺癌有效準(zhǔn)確的檢測(cè)HER2有無(wú)擴(kuò)增是臨床應(yīng)用Heceptin的絕對(duì)必要條件，也是成功進(jìn)行靶向治療的前題和關(guān)鍵胃癌HER2陽(yáng)性率10%～38%胃癌EGFR表達(dá)陽(yáng)性率42%-77.1%（IHC）EGFR基因熱點(diǎn)位點(diǎn)突變罕見(jiàn)：0%-2.6%Kras突變率0-10%Braf突變率0-2.3%PIK3CA突變率6%胃癌基因表達(dá)和相關(guān)基因的突變胃癌HER2異質(zhì)性表達(dá)：部位異質(zhì)性：胃食管結(jié)合部癌(32%)高于胃體(20.9%)類型異質(zhì)性：腸型(32.1%)胃癌高于彌漫型胃癌(6%)腫瘤組織內(nèi)異質(zhì)性MSI/MMR：腸癌預(yù)后及5FU療效預(yù)測(cè)微衛(wèi)星是一種短串聯(lián)重復(fù)序列即DNA重復(fù)序列，以1～6個(gè)核苷酸為一個(gè)單位重復(fù)10～60次。

MSI

（microsatellite

instability，微衛(wèi)星不穩(wěn)定性）：由于重復(fù)單位的插入或缺失

而造成的微衛(wèi)星長(zhǎng)度的任何改變，出現(xiàn)新的微衛(wèi)星等位基因。MSI由MMR

（mismatch

repair，錯(cuò)配修復(fù)基因）缺陷造成。MMR基因（主要是MLH1、MSH2、PMS2、MSH6）失去功能，導(dǎo)致不能修復(fù)DNA復(fù)制過(guò)程中出現(xiàn)的錯(cuò)配，進(jìn)而產(chǎn)生MSI表型。MSI-H（高度微衛(wèi)星不穩(wěn)定）見(jiàn)于90%的林奇綜合征和10%~15%的散發(fā)性結(jié)直腸癌11.

Expert

Rev

Gastroenterol

Hepatol,2011,5(3):385-399.MSI結(jié)直腸癌的病理特征(散發(fā)性結(jié)直腸癌)①散發(fā)性結(jié)直腸癌多位于近端結(jié)腸，易伴發(fā)腸內(nèi)或腸外其他器官的多發(fā)性腫瘤②大約15％的散發(fā)性結(jié)直腸癌顯示MsI③較少發(fā)生淋巴結(jié)轉(zhuǎn)移，生物學(xué)行為較好④與MSS比較，MSI腫瘤復(fù)發(fā)率低⑤腫瘤細(xì)胞DNA多為二倍體或近二倍體⑥其病因通常是hMLHl基因啟動(dòng)子甲基化，MMR基因沉默導(dǎo)致免疫組化顯示相關(guān)MMR蛋白陰性，表現(xiàn)MSI⑦對(duì)某些化療藥物(如5Fu、順鉑等)有原發(fā)性耐藥⑧免疫組化檢測(cè)的靈敏度與特異度分別為83%和88.8%，而

MSI檢測(cè)為89%和90%PRIME（2013）：從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分析結(jié)果，EMA（歐洲藥監(jiān)局）于2013年6月27日發(fā)布帕尼單抗適應(yīng)癥修改信息：僅用于RAS野生型患者

基于OPUS研究Biomarker分析結(jié)果，EMA于2013年11月21日發(fā)布西妥昔單抗適應(yīng)癥修改信息：僅用于RAS野生型患者

規(guī)定對(duì)RAS突變狀態(tài)的檢測(cè)應(yīng)包括KRAS(exons2,3,and

4)和NRAS(exons

2,3,and

4)Biomarker檢測(cè)：推動(dòng)治療方案發(fā)展BRAF：突變預(yù)后更差CRYSTAL/OPUS綜合分析提示：BRAF突變是預(yù)后不良因素之一，但不能預(yù)測(cè)西妥昔單抗療效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突變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時(shí)間(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個(gè)外顯子(圖1),共用2～5外顯子,編碼UGT1A蛋白共同C末端,是尿苷二磷酸葡萄糖醛酸(UDPGA)結(jié)合域,共有246個(gè)氨基酸殘端,組成了UGT1A的保守序列;其余13個(gè)外顯子,均為第1外顯子,序列有37%～90%的同源性,編碼UGT1A蛋白特異的N端,有285個(gè)氨基酸殘基,是UGT1A的底物識(shí)別區(qū)。13個(gè)第1外顯子共編碼9種功能性UGT1A

蛋白(UGT1A1、UGT1A3、UGT1A4、UGT1A5、UGT1A6、UGT1A7、UGT1A8、UGT1A9、UGT1A10)和4個(gè)假基因(UGT1A12P、UGT1A11P、UGT1A13P、UGT1A2P)UGT1A1是一種什么酶尿苷二磷酸葡糖醛酰轉(zhuǎn)移酶（UGTs)是一大類能催化葡萄糖醛酸與親核底物結(jié)合的膜結(jié)合酶，主要存在于肝臟人類的UGTs被分為UGT1，UGT2兩個(gè)家族UGT1的基因至少包括13個(gè)亞型：UGT1A1，UGT1A3-10，UGT1A12P、UGT1A11P、UGT1A13P、UGT1A2PUGT1A1，降解伊立替康。A（腺嘌呤）、T（胸腺嘧啶）、G（鳥(niǎo)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啟動(dòng)子區(qū)EXONGenet

Med

2009:11(1):21–34不同人群的發(fā)生基因型或突變概率略有不同！c-kit/PDGFRA突變類型預(yù)測(cè)伊馬替尼療效，其中c-kit外顯子11突變療效最佳PDGFRA

D842V突變者對(duì)伊馬替尼原發(fā)耐藥。檢測(cè)方法：DNA測(cè)序胃腸道間質(zhì)瘤與格列衛(wèi)基因重排基因重排基因重排對(duì)淋巴瘤診斷的意義確定淋巴組織增生性疾病的克隆性 區(qū)分淋巴瘤和反應(yīng)性增生區(qū)別腫瘤性B和T細(xì)胞檢查微小殘余灶確定淋巴瘤細(xì)胞的來(lái)源幾乎所有的B細(xì)胞淋巴瘤表現(xiàn)出Ig重鏈和輕鏈基因單克隆性重排大多數(shù)T細(xì)胞淋巴瘤顯示TCR基因單克隆性重排淋巴瘤相關(guān)分子診斷BCL6基因斷裂檢測(cè)意義（雙色分離探針）BCL6基因重排的檢測(cè)可以輔助診斷彌漫性大B細(xì)胞淋巴瘤。BCL6陽(yáng)性患者診斷治療36個(gè)月后，疾病停止發(fā)展的比率為82%，攜帶有BCL6重排的病例預(yù)后較好。C-MYC基因斷裂檢測(cè)意義（雙色分離探針）約80%的伯基特淋巴瘤病例發(fā)生t(8；14)（q24;q32）；約15%的伯基特淋巴瘤病例發(fā)生t(2；8)(p11;q24)；約5%發(fā)生t(8；22)（q24;q11）。C-MYC基因斷裂重組可能是伯基特淋巴瘤的一個(gè)標(biāo)志，可以應(yīng)用于臨床上伯基特淋巴瘤的輔助診斷BCL2/IGH融合基因檢測(cè)意義（雙色雙融合探針）輔助診斷濾泡性淋巴瘤BCL2/IGH持續(xù)陰性的FL患者3年生存率為100%，而陽(yáng)性者3年生存率只有54