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Clinical and Biological Insights into Stereotactic Ablative Radiotherapy

Molecular and Clinical Radiobiology Workshop

McGill University Health Centre, June 17-19, 2015

Bassam Abdulkarim, MD, PhD Associate Professor, Department of Oncology,

Division of Radiation Oncology, McGill University Cancer Research Program

Research Institute of the McGill University Health Centre

bassam.adulkarim@mcgill.ca

Early&stage+NSCLC&Lung%cancer%is%the%most%frequent%cause%of%cancer%related%death%in%males%and%second%in%females.%%%%%%%%%Non6small%cell%lung%cancer%(NSCLC):%75680%%%of%all%lung%cancer%Early%stage%NSCLC:%20625%%of%all%NSCLC%The&combina8on&of&popula8on&aging&and&oncoming&CT+based&screenings&programs&will&increase&the&number&of&diagnosed&early&stage&lung&cancer&especially&in&the&elderly&pa8ents&% Smith&BD.&J&Clin&Oncol&2009;27:2758965.&&

Na=onal&Lung&Screening&Trial&Research&Team.&N&Engl&J&Med&2011;365:3959409.&&

Stage&shit&from&advanced&stage&to&a&stage&I&&

Op8ons&of&Early&stage+NSCLC&

&Surgery&%6%Lobectomy%%6Wedge%resecFon%%6Video6assisted%thoracoscopy%surgery%(VATS)%%

%Radia8on&Therapy&

%6%ConvenFonal%RT%%6%Stereotac8c&Body&Radia8on&Therapy&(SBRT)&&

%Radiofrequency&Abla8on&%%%%%

Early&stage+NSCLC&%About%80%%of%stage%I%disease%paFents%undergo%surgical%resecFon(1)%PaFents%with%comorbidity:%sublobar%resecFon%(2).%%%Elderly%paFents%with%increasing%comorbidiFes:%will%not%receive%a%curaFve%local%treatment%(up%to%26%)%(3)%%In%the%USA:%decline%in%the%use%of%surgical%resecFon%from%75%%to%63%%despite%the%increasing%use%of%less%invasive%surgery,%Video6assisted%thoracoscopy%surgery%(VATS).%%Data%from%Amsterdam:%this%percentage%drop%under%40%%for%paFents%>75%years%%%19Whitson&BA.&Ann&Thorac&Surg&2011;92:1943950.&29Raz&DJ.&Chest&2007;132:19399&&&39De&Perrot&M.&Eur&Respir&J&1999;14:419922.&&

For%inoperable%paFents,%convenFonal%RT%:%60666%Gy%in%2%Gy6fracFons%over%a%Fme%period%of%667%weeks.%Overall%survival%(OS)%of%about%30%%at%3%years%(1)%%Local&tumor&relapse&being&the&most&frequent&site&of&failure&(2)%Non6operable%paFents:%introducFon%of%novel%radiotherapy%concepts%and%technologies:%Stereotac8c&Abla8ve&Radiotherapy&(SABR)&%%

%6%Combines%several%modern%technologies%to%accurately%treat% %%%%%tumors%with%very%high%radiaFon%doses.%

%%%6%RT%doses%are%delivered%in%few%fracFons%or%even%in%one%%%%radiosurgical%session.%

%%%%

19&DeQerbeck&FC.&J&Thorac&Oncol&2008;3:781992&29&Rowell&NP.&Thorax&2001;56:628938.&.&&

Early&stage+NSCLC&

Outline&

I6%Early%stage%non%small%cell%lung%cancer%management%with%SABR%%II6%Biology%of%SABR%in%non%small%cell%lung%cancer%%III6%4%(5)%R’s%of%radiobiology%and%Linear%QuadraFc%model%for%SABR%%IV6%RelaFonship%between%radosensiFvity%and%invasion/metastasis%in%context%of%SABR%V6%Conclusions%%%%

I+&Early&stage&non&small&cell&lung&cancer&management&with&SABR&%II6%Biology%of%SABR%in%non%small%cell%lung%cancer%%III6%4%(5)%R’s%of%radiobiology%and%Linear%QuadraFc%model%for%SABR%%IV6%RelaFonship%between%radosensiFvity%and%invasion/metastasis%in%context%of%SABR%%%%

Contents&

Elements&of&SABR&

ES+NSCLC&management&with&SABR&&

19&Onishi&H.&J&Thorac&Oncol&2007;2:S94–S100.&29&Olsen&JR.&Int&J&Radiat&Oncol&Biol&Phys&2011;81:e299–303.&39&Zhang&J.&Int&J&Radiat&Oncol&Biol&Phys&2011;81:e305–16.&

SABR:%accurate%and%precise%delivery%of%high%doses%of%radiaFon%over%a%few%fracFons,%%%%%RadiaFon%doses%that%are%biologically%equivalent%to%100%Gy10%or%more%are%required%to%achieve%high%local%control%rates%(1,%2).%%%These%doses%are%in%excess%of%what%is%pracFcal%using%convenFonally%fracFonated%treatment%schemes.%%OpFmal%SABR%dose%is%unknown,%although%a%meta6analysis%suggests%that%highest%(>146%Gy10)%BED%fracFonaFons%may%have%lower%OS%than%medium–high%(106–146%Gy10)%fracFonaFons%[3].%%%

Onishi&H.&J&Thorac&Oncol&2007;2:S94–S100%

local%control%and%5%years%survival%rates%were%beder%with%a%BED%of%100%Gy%or%more%compared%with%less%than%100%Gy.%%

ES+NSCLC&management&with&SABR&&

IntroducFon%of%SABR%in%elderly%paFents%was%associated%%6%Absolute%increase%of%16%%in%RT%use%%6%Decline%in%the%proporFon%of%untreated%paFents%and%improvement%%in%OS%(1).%%

%56year%overall%survival%of%paFents%with%stage%I%NSCLC%treated%with%SABR%might%be%equivalent%to%surgery,%even%in%operable%paFents%%%%Phase%III%studies%(ROSEL,&STARS,&RTOG&1021)%launched%between%2008%and%2011%and%opened%at%77%centres%were%closed%for%poor%accrual.%%%%%%

16%Palma%D.%J%Clin%Oncol%2010;28:5153–9%26%FJ%Lagerwaard.%Int%J%Radiat%Oncol%Biol%Phys,%83%(2012),%pp.%348–353%

Reported%

Operable&Stage&I&NSCLC+&SABR&trials&&

ES+NSCLC&management&with&SABR&%%Nonetheless,%development%of%their%protocols%provided%the%radiaFon%oncology%community%with%valuable%standards%in%quality6assurance%and%for%broad%implementaFon%of%lung%SABR%%In%the%absence%of%RCT%data,%other%forms%of%ComparaFve%effecFveness%Research%(CER)%are%crucial%in%guiding%decision%making%%%CER%studies%using%propensity6score%matching,%match6pair%analysis,%Markov%modeling,%cost6effecFveness%and%meta6analysis%methodologies.%

Louie&AV&et&al&.&Radiotherapy&and&Oncology&114&(2015)&138–147&

Compara8ve&effec8veness&Research&&to&Guide&Clinical&Prac8ce&

The%absence%of%randomized%data%does%not%imply%the%absence%of%evidence.%CER%is%to%inform%clinical%pracFce%%%56year%OS%with%stage%I%non6small6cell%lung%cancer%(NSCLC)%given%SABR%might%be%equivalent%to%surgery,%even%in%operable%paFents%fit%to%undergo%resecFon%(1,%2)%%A%propensity%score6matched%analysis%using%the%SEER6Medicare%database%suggests%that%short6term%mortality%is%improved%with%SABR%(<1%)%compared%with%either%lobectomy%or%sublobar%resecFon%(about%4%),%and%long6term%survival%did%not%differ%(3).%%%%19&FJ&Lagerwaard,&&Int&J&Radiat&Oncol&Biol&Phys,&83&(2012)&29&H&Onishi,&Int&J&Radiat&Oncol&Biol&Phys,&81&(2011)&39&S&Senthi,&&Radiother&Oncol,&106&(2013)&

&

Experience:%tumors%>5%cm%can%be%safely%and%effecFvely%treated%with%SABR%if%dose%constraints%to%criFcal%structures%as%suggested%by%Timmerman%are%respected%(1).%%%SABR%for%T2:%associated%with%nodal%and%distant%failure%and%trended%toward%a%worse%survival%than%did%T1%lesions%(2)%%Tumor%size%is%an%important%predictor%of%response%to%SABR%and%should%be%considered%in%treatment%planning.%%19Timmerman&RD.&Semin&Radiat&Oncol&18:2159222,&2008&29&Dunlap&NE.&1,&J&Thorac&Cardiovasc&Surg.&2010&&&&

&Should&Larger&Tumors&Be&Treated&With&SABR?&

&

PaPerns&of&recurrence&aQer&SABR&

PaPerns&of&recurrence&in&RTOG&0236:&the%predominant%padern%of%recurrence:%was%regional%and%distant%recurrence%&56year%primary%tumor%failure%rate%was%7%%%56year%local6regional%failure%rate%was%38%%%56year%rate%of%distant%failures:%31%%%%

%%%%%%%

Timmerman%R.%Int%J%Radiat%Oncol%BiolPhys%90:S30,%2014%%

The%predominant%paderns%of%recurrence%was%distant%recurrence%% %6%56year%rate%of%local%failures:%10%%

%6%56year%rate%of%regional%failures:%12%%%6%56year%rate%of%distant%failures:%20%%

6 %Isolated%distant%recurrence%accounted%for%46%%of%all%recurrences%and%occurred%at%a%median%of%8.3%months%amer%SABR%6 PaFents%with%inoperable%stage%I%NSCLC%treated%with%SBRT%had%a%survival%rate%of%40%%at%5%years.%%%%

SABR&impact&in&ES+NSCLC&IntroducFon%of%SABR%in%elderly%paFents%was%associated%%

%6%Absolute%increase%of%RT%use%by%16%%%6%Improvement&of&5+year&overall&survival&%%%6%Propensity%matched%score%analysis:%survival%of%paFents%with%%%stage%I%given%SABR%might%be%equivalent%to%surgery%

%

SABR&and&Risk&Adap8ve&Regimen:&&%6%AdapFve%dose%and%schedule%based%on%tumor%locaFon%%6%No%adapFve%dosing%based%on%T%size%%6%No%associaFon%between%SABR%and%GeneFc%AlteraFon%such%as:%%%%EGFR,%ALK,%K6Ras%

The&predominant&paPerns&of&recurrence&is&distant&recurrence:&20+30%&

Contents&

I6%Early%stage%non%small%cell%lung%cancer%management%with%SABR%%II+&Biology&of&SABR&in&non&small&cell&lung&cancer&%+&4&(5)&R’s&of&radiobiology&and&Linear&Quadra8c&model&for&SABR&%6%RelaFonship%between%radiosensiFvity%and%invasion/metastasis%in%context%of%SABR%%%%

4&(5)&R’s&of&Conven8onal&Frac8onated&RT&

SABR&and&redistribu8on/&repopula8on&

%%&

SABR%→%high%levels%of%DNA%damage,%repair%evident%@%80%Gy%No%evidence%of%repair%saturaFon%%(1)%%%High6dose%radiaFon6induced%foci%(RIF)%formed%relaFvely%faster%and%resolved%slower%than%low6dose%RIF%(1)&&%

%

SABR&and&Repair&

Carlson%et&al:&in&hypoxic&situa=ons!(1)!!3!logs!of!cell!kill!lost!up!to!single!doses!to!18524!Gy!!Can%be%overcome%with%hypoxia%dose%boosFng&(2,3)&%Brown%et&al.&(2010)&evaluated&the&expected&level&of&radia=on9mediated&cell&killing&by&different&SBRT&regimens!20!Gy!x!3!was!barely!sufficient!due!to!hypoxia!(4)!%Clinical%outcomes%for%NSCLC%with%SABR%are%good%IndicaFve%of%mechanisms%in!addiBon!to!direct!cell!killing!!AnF6tumor%immune%responses,%secondary%effects%from%vascular%damage%%%%

Reoxygena8on&(hypoxia)&and&SABR&%

Hypothe8cal&cell&death&mechanisms&aQer&SABR&

(1) Direct&and&indirect&vascular&damage%6 %Large%fracFon%10%Gy%or%higher%may%prohibit% %%reoxygenaFon%and%hypoxic%tumor%cells%(1)%

6 Decrease%in%viability%of%tumor%cells%over%2%days%amer%single%dose%of%10%Gy%in%Rat%model%due%to%vascular%damage%

16%Clement%JJ,%Takanka%N,%Song%CW.%Radiology%1978;127:7996803.%

Poten8al&mechanisms&for&IR&effects&on&vasculature&

•  InducFon%of%endothelial6cell%apoptosis%•  AlteraFon%of%angiogenic%factors%and%cells%in%the%microenvironment%–  VEGF%–  CirculaFng%CD11b+%cells%

Vasculature&–&important&determinant&of&tumor&radioresponse&with&abla8ve&RT?&

Tumors have marked resistance to single high dose radiation (15 Gy) when endothelial apoptosis is inhibited *This remains an area of controversy*

Science 2003

%(2)&Indirect&effects:&Cancer&stem&cells%6 %Cancer%stem%cells%are%considered%radioresistant%(1)%6 %Cancer%stem%cells%idenFfied%in%perivascular%niche%(2)%6 SBRT%destroying%endothelial%cells%may%eradicate%cancer%stem%cells%

&&Hypothe8cal&cell&death&mechanisms&aQer&SABR&

%(3)&Indirect&Effects:&An8+tumor&&immunity%6 SBRT%may%turn%the%tumor%into%an%“immunogenic&hub”:%priming%systemic%immune%response%6 %Clinical%evidence%SBRT%contributes%to%anFtumor%immunologic%at%a%distant%site%(2)%

Effect of dose and fractionation on immune response

As lymphocytes are sensitive to RT: repetitive daily delivery can deplete migrating immune effector cells. Single dose (12 Gy) RT did not deplete established tumors from immune effector cells (CD8+ T, CD4+ T and NK cells) critical to the curative activity of RT when used in combination with immunotherapy. Indeed, irradiated mammary tumors were enriched for functionally active, tumor-specific T cells and Ly-6C+ memory CD8+ T cells I. Verbrugge, Cancer Res. 2012 S.E. Finkelstein Clin. Dev. Immunol. 2011

Effect of dose and fractionation on immune response

The spectrum of dose used in SABR: greater augmentation of the tumoricidal immune response than conventional radiotherapy

- Greater degree of stromal/vascular damage, ceramide-induced endothelial cell damage and increased apoptosis of tumor cells

- Cross-priming of T-cells against tumor antigens have been demonstrated to be induced by a single dose of 15 Gy. This effect was significantly inferior in mice treated with a 3 Gy x 5 fractions regimen I. Verbrugge, Cancer Res. 2012 S.E. Finkelstein Clin. Dev. Immunol. 2011

Mechanisms of abscopal effects Inhibitory role for local radiation on distant tumour growth (R J Mole, Br J Radiol 1953) Abscopal effect after radiotherapy has been detected in lymphoma, melanoma, and a variety of carcinomas.

Inflammatory signaling and the immune system have been recognized as key components of transmission of abscopal effects Two opposite clinical strategies: (1)  Induce the abscopal remission of metastatic tumors by

acute immunological activation with ionizing radiation (2) Searching for messenger molecules that could be a therapeutic target for limiting radiation-induced cancers.

%High6dose%fracFon%(HDF)%SABR%Not%well6described%by%LQ%%Disconnect%between%LQ%model%and%SABR%

%6%Vascular%effects%amer%HDF%SABR%%6%PotenFal%of%immune%effects%by%SABR%%%6%PotenFal%for%stem%cells%eradicated%by%SABR%%

%•Modest%dose%SBRT%(<%8%Gy):%experimental%evidence%tends%to%indicates%LQ%model%is%sufficient%%%%•4%R�s%of%radiobiology:%Re6oxgyenaFon%most%relevant%for%SABR%

Conclusion:&SABR&and&LQ&model&

Contents&

I6%Early%stage%non%small%cell%lung%cancer%management%with%SABR%%II+&Biology&of&SABR&in&non&small&cell&lung&cancer&%+&4&(5)&R’s&of&radiobiology&and&Linear&Quadra8c&model&for&SABR&%IV+&Rela8onship&between&radosensi8vity&and&invasion/metastasis&in&context&of&SABR&%%%

Rela8onship&between&Radosensi8vity&and&Invasion/Metastasis&in&context&of&SABR&

Specific&Objec8ves&

•  Compare%response%of%NSCLC%cell%lines%to%AblaFve%(ART)%and%fracFonated%(FRT).%

•  Response%to%ART%based%on%geneFc%alteraFons%such%as%EGFR%and%K6Ras.%

•  ValidaFon%of%in&vitro&findings%in%an%orthotopic%NSCLC%animal%model.%

Adenocarcinoma&Cell&lines&

Cell&Line& p53& EGFR& K+RAS& SF2&(Radioresistance)&

A549% WT% WT%(+)% Mut(G12S)%%

0.74%%(++)%

HCC827% p.V218del% Del%E7226746% WT% 0.67%(+)%

H1975% WT% L858R/T790M% WT% 0.34%(6)%

CELLULAR&RESPONSE&OF&NSCLC&CELLS&TO&ABLATIVE&AND&FRACTIONATED&RT&

Assays&&&Endpoints&

Clonogenic%PotenFal%

ProliferaFon:%MTT%

Invasion:%Matrigel%Assay%

Cell%Death/%Arrest%

Morphological Assessment

0 2 4 6 8 100.0001

0.0010

0.0100

0.1000

1

Dose (Gy)

Su

rviv

ing

Fra

ctio

n

Curve: Nonlin fit of Row Stats of GroupCurves

A549HCC827H1975

0 2 4 6 8 100.0001

0.0010

0.0100

0.1000

1

Dose (Gy)

Sur

vivi

ng F

ract

ion

Curve: Nonlin fit of Row Stats of GroupCurves

A549HCC827H1975

A

8Gy

12Gy

0

20

40

60

80

Dose

% P

rolif

erat

ion

A549

ARTFRT

A549

*** *** B

1 10

20

40

60

80

Dose

% P

rolif

erat

ion

A549

AIRFIR

8Gy

12Gy

0

20

40

60

80

Dose

% P

rolif

erat

ion

H1975

ARTFRTH1975

8Gy

12Gy

0

20

40

60

80

Dose

% P

rolif

erat

ion

HCC827

ARTFRT

C D

HCC827

0 24 48 72 96 1200

20

40

60

80

Time Post Irradiation (hours)

% C

ell D

eath

A549 Death

0Gy12Gy

0 24 48 72 96 1200

20

40

60

80

Time Post Irradiation (hours)

% C

ell D

eath

HCC827 Death

0Gy12Gy

0 24 48 72 96 1200

20

40

60

80

Time Post Irradiation (hours)

% C

ell D

eath

H1975 Death

0Gy12Gy

A549 HCC827 H1975

0 24 48 72 96 1200

20

40

60

80

Time Post Irradiation (hours)

% C

ell D

eath

H1975 Death

0Gy12Gy

Cell%Death/Morphology%

Untreated( 12Gy(4x3Gy(

A549(

HCC827(

H1975(

Untreated( 12Gy(4x3Gy(

A549(

HCC827(

H1975(

A549&cells&forma8on&of&numerous&cell&protrusions&and&the&predominance&of&polynucleated&cells&&&

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

AP: 0.8% G1:51.5% S: 35.4% G2:10.8%

A549

AP: 1.4% G1:45.5% S: 5.0% G2:47.2%.

AP: 2.8% G1:33.6% S: 26.7% G2:29.1%

AP:11.6% G1:74.7% S: 13.5% G2: 0.0%

HCC827

Control IR-12Gy

AAAAP A

AP: apoptosis

AP:16.9% G1:60.4% S: 8.1% G2:13.7%

AP: 0.3% G1:43.3% S: 18.3% G2:27.6%

H1975

Cell%cycle%and%Apoptosis%

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

AP: 0.8% G1:51.5% S: 35.4% G2:10.8%

A549

AP: 1.4% G1:45.5% S: 5.0% G2:47.2%.

AP: 2.8% G1:33.6% S: 26.7% G2:29.1%

AP:11.6% G1:74.7% S: 13.5% G2: 0.0%

HCC827

Control IR-12Gy

AAAAP A

AP: apoptosis

AP:16.9% G1:60.4% S: 8.1% G2:13.7%

AP: 0.3% G1:43.3% S: 18.3% G2:27.6%

H1975

Cell%cycle%and%Apoptosis%

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

PARP$

p53$

p21$

β+ac.n$

0Gy$ 12Gy$

AP: 0.8% G1:51.5% S: 35.4% G2:10.8%

A549

AP: 1.4% G1:45.5% S: 5.0% G2:47.2%.

AP: 2.8% G1:33.6% S: 26.7% G2:29.1%

AP:11.6% G1:74.7% S: 13.5% G2: 0.0%

HCC827

Control IR-12Gy

AAAAP A

AP: apoptosis

AP:16.9% G1:60.4% S: 8.1% G2:13.7%

AP: 0.3% G1:43.3% S: 18.3% G2:27.6%

H1975

Cell%cycle%and%Apoptosis%

0Gy$ 4x3Gy$ 12Gy$

A549$

H1975$

HCC827$

Matrigel&Invasion&

!"#$%#&''#()*'+!',-./012'34..'35.6574'"18476

+!',-./01'"18476'984:';<6='-'+!',-./01'30>?-1<01'(<8854'35.6574'@.-64

+!',-./01'34..'35.6574'"18476

!"#$%&

'&()*#$&

+,&-./-!#"01$-2&""-23"43*&-5$6&*421(7#$81$+2-9"#4&-:&#43*&6-#-1$&;<#=>--"#)=*8$4,"8?-?&68%$-#$?-01$?&$6#481$-*8$%6-41(8$8(8@&-&A#71*#481$-#$?-01$4#(8$#481$B

AB',C%'"D39+#("CD----C"8?&-4,&-8$6&*4-)#0D-8$41----4,&-<&"">--7"#0&-4,&----:"#$%&6-8$-4,&-$140,&6B----+,86-<8""-(8$8(8@&-(&?8#----<80D8$%-#$?-#663*&----7*17&*-7"#4&;"8?-:84B

EBCB-9#4&$4-F1B-G>HII>JKH-#$?-14,&*-9#4&$46-9&$?8$%

D06/=48

+!',-./012'3C&@#D"CD'(3'@E#(*./-2#4B-F1B-HGHGLMN--I-<&""6./-2#4B-F1B-HGHGLHN--OM-<&""6./-2#4B-F1B-HGHGLPN--MP-<&""6

FB',C%',**!"D$- C"8?&-4,&-8$6&*4-41-4,&

68?&-1:-4,&-<&""-:1*-&#6=787&4-#00&66B

Matrigel%Layer%0.8uM%Pores%

Boyden%Chamber%

50,000%Cells%(starved)%

•  ART&increased&invasion&in&A549&cells&only.&&

•  ART&and&FRT&reduced&invasion&equally&in&&HCC827&and&H1975&cells.&

0Gy

12Gy

0Gy

12Gy

0.0

0.5

1.0

1.5

2.0

Dose (Gy)

Fold

Diff

eren

ce

A549 Invasion

InvasionMigration

0Gy

12Gy

0Gy

12Gy

0.0

0.5

1.0

1.5

2.0

Dose (Gy)

Fold

Diff

eren

ce

HCC827 Invasion

InvasionMigration

0Gy

12Gy

0Gy

12Gy

0.0

0.5

1.0

1.5

2.0

Dose (Gy)

Fold

Diff

eren

ce

H1975 Invasion

InvasionMigration

0Gy

12Gy

0Gy

12Gy

0.0

0.5

1.0

1.5

2.0

Dose (Gy)

Fold

Diff

eren

ce

HCC827 Invasion

InvasionMigration

Cell%Invasion%and%MigraFon%

A549%cells%exposed%to%ART,%%showed%a%significant%increase%(26fold)%in%cell%migraFon%and%invasion%compared%to%untreated%cells.%%%HCC827%cells%exposed%to%either%ART%showed%significantly%lower%migraFon%and%invasion%compared%to%untreated%cells%%H1975%cells,%ART%did%not%significantly%impact%migraFon%levels,%but%significantly%reduced%invasion%

A549& HCC827& H1975&

•  ART%differenFally%impacts%cellular%response%in%NSCLC%cell%lines%compared%to%FRT%in%A549%cells%–  Cell6line%dependent?%

•  In%HCC827%&%H1975%cells%–  FRT%and%ART%induced%similar%inhibiFon%of%proliferaFon%and%phenotypic%changes.%

–  FRT%and%ART%equally%reduced%invasion.%

Conclusions%

•  In%A549%cells%–  Cell%proliferaFon%is%significantly%inhibited%with%ART%compared%to%FRT.%

–  Cell%morphology%and%size%are%altered%amer%ART%compared%to%FRT.%

–  ART%resulted%in%significant%increase%in%invasion%

Conclusions%

PATHWAYS&AND&PROTEINS&REGULATING&IR+INDUCED&CELLULAR&CHANGES&

Understand%the%underlying%biology%for%increased%inhibiFon%of%proliferaFon%and%increased%invasion%amer%ART%in%A549&cells.&

ART&differen8ally&impacts&cellular&response&based&on&gene8c&altera8ons&&&

Feature/Response& A549& HCC827& H1975&

p53% WT% p.V218del% WT%

EGFR% WT%(+)% Del%E7226746% L858R/T790M%

K6RAS% Mut(G12S)% WT% WT%

SF2% 0.76% 0.64% 0.35%

Cell%Death% None% High% High%

Apoptosis% Non% Increased% Increased%

ProliferaFon% Reduced% Halted% Halted%

Invasion% Increased% Reduced% Reduced%

Role&of&EGRF,&and&K+Ras&in&ART&response&6 A549%with%EGFR%mutaFon%(L858R%and%Del%7226746)%6 A549%with%K6Ras%funcFon%restored6Dr%A.%Nepveu%at%GCC%

K+Ras&muta8on&and&&Radioresistance&

EGFR%mutaFon%and%%RadiosensiFvity%

In%Vivo%Study%

•  Orthotopic&NSCLC&•  1x106&A549&cells&in&matrigel&•  CT+guided&i.c.&injec8on&(ribs&5+6)&&•  4&weeks&for&tumor&growth&&

&&imaging,&then&randomiza8on&•  2&Groups&of&animals&

–  ART&GROUP&(6)&–  Control&GROUP&(4)&

Animal%Study%(AUP%#7063)%

CT of rat

Histochem Cell Biol (2008) 130:481–494 487

123

Oncochip microarray of metastatic versus non-metastaticmelanomas identiWed diVerentially expressed genes impli-cated in EMT. Validation of these expression data in anindependent series of melanomas using tissue microarraysconWrmed that the expression of a set of proteins includedin the EMT group (N-cadherin, osteopontin and SPARC)was signiWcantly associated with metastasis development(Alonso et al. 2007). A similar oligonucleotide microarraystudy on papillary thyroid carcinoma from central and inva-sive regions and of normal thyroid tissue was performed.The invasion fronts were consistently characterized by thepresence of mesenchymal markers and absence of epithelialmarkers. Furthermore, immunohistochemical analysisrevealed that overexpression of vimentin associated withpapillary thyroid carcinoma cell invasion (Vasko et al.2007). Molecular classiWcation of fresh-frozen and forma-lin-Wxed head and neck squamous cell carcinomas showedthat genes involved in EMT are the most prominent mole-cular characteristics of high-risk tumors (Chung et al.2006). In conclusion, we strongly insist that the occurrenceof the EMT signature should be explored in large-scalestudies of human clinical tumors in order to predict theirmetastatic potential.

Pathological signatures of EMT in biological Xuids

A number of molecules related to EMT can be assessed inbiological Xuids from cancer patients. Particularly in serumthey reXect either the pathogenesis or the consequences ofthe phenomenon. Table 1 summarizes their applications indiVerent types of cancer.

Scatter factor/hepatocyte growth factor concentrations inserum are correlated with metastatic spread, they possessprognostic value, and they are useful for monitoring ther-apy. They can also be used as a urine parameter for diagno-sis of transitional carcinoma of the bladder. Fibroblast

growth factor-2 (FGF-2), another tyrosine kinase receptorligand, is an endothelial-to-mesenchymal transformationmarker, its serum concentrations correlate with tumorgrowth rate, volume, grading and staging. Its main clinicalusefulness, however, resides in monitoring the eVect ofangiogenesis inhibitors. In prostatic cancer an inverse cor-relation between prostate-speciWc antigen (PSA) and FGF-2 has repeatedly been observed (Usami et al. 2008). Whilethe EMT inducer TGF-! as a circulating marker is diYcultto interprete, the serum concentrations of its functional acti-vators, MMP-9 and CD44 (mainly its spliced isoform v6),have proven to possess prognostic information. Assessmentof the urine concentrations of MMP-9 was claimed to con-tribute to the diagnosis of bladder cancer. Circulating tenas-cin-C, a matricellular protein acting in concert with SF/HGF, can discriminate between good and bad prognosis innumerous cancers, and its cut-oV level was determined at96 ng/ml. Tenascin-C serum concentrations also correlatedwith the vascularization of the tumors. A central player incancer progression is p53, and this tumor suppressor’s levelin circulation was found to be higher in cancer patients thanin normal individuals. This is presumably the result of theincreased biological half-life of many p53 mutants. More-over, mutations in exons 5 and 6 are particularly immuno-genic, and elicit detectable auto-immune anti-p53antibodies in the circulation (IgM and IgG). Although p53and its antibodies were shown to correlate with prognosisand therapeutic response of head- and neck cancer, theirmajor applicability is in epidemiological studies, wheremutagenic pressure from the environment has to beassessed.

The switch from E- to N-cadherin is considered typicalfor EMT, and enzymatically shed, soluble ectodomainsfrom both cadherins have been detected at elevated levelsin the serum of cancer patients reviewed in (De Weveret al. 2007). Conceptually the measurement of solubleE-cadherin (sE-cad) may be less straightforward, because

Fig. 3 Histology of basal cell carcinoma. Hematoxylin and eosinstaining of a basal cell carcinoma showing the collective invasion ofepithelial cancer cells into the extracellular matrix (arrows, panel A).Single cancer cells detached from the tumor mass and showing traits of

epithelial–mesenchymal transition are indicated by arrows (panel B).Stromal (myo)Wbroblasts are indicated by arrowheads. Scalebars = 50 !m

Cell&Inocula8on& • Day%0%

Weekly&Imaging& • D14%–%D21%

Radiotherapy& • D21%–%D30%• 34Gy%vs%0Gy%

Histology&&&IHC&

• D40%–%D100%• Euthanize%

•  Serial&CT&Imaging:&&–  Tumor%growth%and%local%control%–  Regional/Distant%metastasis%(lung,%lymph%nodes,%brain,%liver,%kidneys/adrenals,%spleen)%

•  Histopathology&–  Tumor%type%and%differenFaFon%(mucin,%polynucleaFon)%–  Extent%of%invasion%–  Cellular%characterizaFon%and%morphology%–  MitoFc%index%(#%of%Ms%in%5%HPFs)%

•  IHC&–  EMT%makers:%vimenFn,%E6cadherin,%Beta6catenin,%CK7%–  ProliferaFon:%Ki667%%–  Invasion%markers:%cMET%

Endpoints%and%Assays%

3D%Dose%Max:%100.7%%3D%MAX%for%GTV:%100.7%%3D%MIN%for%GTV:%90.6%%3D%MEAN%for%GTV:%97.7%%

0 15 22 28 35 43 500.0

0.5

1.0

1.5

2.0

0Gy Group

Days

Tum

or V

olum

e (c

m3 )

R12R2R4R24R16

Tumor%Response%

0 15 22 28 35 43 50 60 70 800.0

0.5

1.0

DaysTu

mor

Vol

ume

(cm

3 )

34Gy Group

R13R1R6R10R17R11

Heterogeneity in tumor growth rate Treatment delivery when tumor size > 0.1 cm3

4/6 = CR 1/6 = PR 1/6 = DP

R13 - Complete response

Week 2 Week 3 Week 4

Week 5 Week 6

Radiation-Induced Pneumonitis

0.20cm3

Week 2 Week 3 Week 4

Week 5 Week 6 Week 7

R1 – Tumor progression D2

D4

0.31cm3

34Gy

Week 6

Week 2 Week 3

Week 5

Week 4

Week 7

R17 – Partial Response

0.88cm3

Week 2

Week 6

Week 3 Week 4 Week 5

Week 7 Week 8 Week 10

Week 14

R10 – Complete response

0.10cm3

Rat&10&Week&13&

At&60&days&post+treatment,&large&chest&wall&mass&and&bone&metastasis&&

R&

Rat&10&

Immunohistochemistry 0Gy 34Gy (10d) 34Gy (30d)

Ki-67

E-cadherin

Vimentin

Beta-catenin

CK7

IRS 12 IRS 12 IRS 12

IRS 12 IRS 12 IRS 12

IRS<4 IRS <4 IRS<4

IRS 12 IRS 12 IRS 12

21% 3% 27%

Metastatic tumor are poorly differentiated. No evidence of EMT in the metastatic tumors.

Increased&invasion&capability&of&Lewis&lung&carcinoma&(LLC)&cells&aQer&7.5 Gy.&%&%LLC&cells&were&subcutaneously&injected&into&right&thighs&and&the&resul8ng&tumors&were&irradiated&(10 Gy&×&5):&Induced&forma8on&of&pulmonary&metastasis.&Radia8on&significantly&enhanced&MMP+9&at&both&the&transcrip8onal&and&transla8onal&levels.&%&%Higher+dose&irradia8on&(30 Gy&×&2)&significantly&reduced&the&number&of&pulmonary&metastases.&%%

Conclusion%and%PerspecFves%

End&of&the&Beginning:%SABR%become%the%state%of%art%for%stage%I%lung%cancer%and%offer%promising%results%for%OS%and%toxiciFes.%Move%away%from%local%control%to%assess%survival,%quality%of%life,%and%invesFgate%the%effect%of%SABR%on%Immune%system,%cancer%stem%cell…%%%The%success%of%SABR%has%been%primarily%technology6driven.%ApplicaFon%of%fully&potent&SABR&regimes&is&now&impeded&by&biological&limita8ons.%%%Aspects%of%both%normal%Fssue%and%tumor%response%require%a%further%understanding%in%order%to%augment%the%technology%gains%as%well%as%to%minimize%the%potenFal%harm%%%

It&may&be&appropriate&to&re+examine&the&place&of&hypoxia&and&metastasis&&in&the&context&of&SABR.%%%%%Our%novel%observaFons%of%a%differenFal%cell6type%based%response%to%SABR%underline%the%need&for&molecular&characteriza8on&of&tumors&and&avoid&delivering&sublethal&dose&to&the&tumor.%%%%Combining%SABR%with%targeted%anF6invasive/hypoxic%drugs%may%provide%an%effecFve%regimen%to%reduce%the%risk%of%metastasis%%Use of Radiation for awakening the dormant immune system, a promising challenge to be explored %%

Conclusion%and%PerspecFves%

Acknowledgements%%

&Ayman&Oweida %PhD%Student&Zeinab&Sharifi % %PhD%Student%Jack&Xu % % %Research%Assistant%%%Dr.&Siham&Sabri,%Research%InsFtute%of%McGill%University%Health%Center%%Dr&Jan&Seuntjens,%Medical%Physics,%Unit%Dr&Russell&Ruo,%Medical%Physics%Unit%

&Montreal&General&Hospital&Cancer&Founda8on;&&Research&Ins8tute&of&McGill&University&Health&Center.&

69%

Collaborators:%

Department%of%Pathology:%Dr&Richard&Frazer&