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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
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®imens!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&-:+*&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*-=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®ULATING&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®imes&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&